Oxytocin is often referred to as a ‘love hormone’ because it can be released during activities such as hugging, snuggling, or sex. Reality, of course, can be a bit more complicated. In the brain, oxytocin can have powerful and diverse effects on mood, stress, anxiety, and social interactions. In the body it helps regulate fluid balance, promotes contractions during childbirth, and stimulates the letdown of milk during breastfeeding.

Much of the oxytocin produced in both humans and rodents comes from oxytocin-synthetizing magnocellular neurons located in an area of the brain called the hypothalamus. These very specialized neurons have separate, but overlapping, mechanisms for releasing oxytocin into the brain and into the rest of the body. This means that while certain signals cause the neurons to release oxytocin into the body and the brain at the same time, others can cause them to release the hormone preferentially into the body or the brain.

Sheng et al. wanted to better understand how these different release mechanisms work, and, in particular, to learn more about how release of oxytocin into the brain is regulated. This is important, because when oxytocin is given as a medicine, much of it fails to reach the brain.

A lot of the oxytocin that acts in the brain is released from a specific part of the oxytocin-synthesizing magnocellular neurons called the dendrites. When these neurons are stimulated, calcium enters the dendrites, triggering the release of oxytocin directly into the brain. Sheng et al. used electrical and optical tools on brain tissue extracted from mice to measure how different signals change the amount of calcium that enters the dendrites of oxytocin-synthesizing magnocellular neurons in response to a consistent stimulus.

The results showed that increasing the osmolarity, the amount of water-soluble particles that cannot spontaneously cross the cell membrane, in the liquid surrounding the neurons reduced the amount of calcium that flowed into the dendrites during stimulation. Meanwhile, decreasing osmolarity had the opposite effect. Sheng et al. also found that the influx of calcium induced by stimulating the neurons can be strongly regulated by activating receptors in the dendrites that detect a common molecule in the brain called GABA. This occurs even absent a change in osmolarity.

These results shed light on some of the physiological processes that control the release of oxytocin into the brain. Understanding these processes is a necessary step towards developing new drugs intended to regulate levels of oxytocin in the brain. Such drugs could be useful in the treatment of several types of mental health disorders.

Oxytocin (OT) is a nine amino acid peptide synthesized almost exclusively in hypothalamic neurons of the supraoptic and paraventricular nucleus (SON, PVN). Despite the highly localized nature of OT synthesizing neurons, OT receptors (OTRs) are distributed widely throughout the CNS. Significant evidence indicates an important modulatory role for central oxytocinergic signaling in a wide variety of processes including fear conditioning, stress responding, anxiety-related behaviors, maternal behavior, sexual behavior, and social recognition (e.g. see Bosch et al., 2005; Knobloch et al., 2012; Jurek et al., 2015; Veening et al., 2015; Neumann and Slattery, 2016; Lin et al., 2018; Tan et al., 2019; Valtcheva and Froemke, 2019; Winter and Jurek, 2019). Numerous studies have demonstrated effects of OTR agonists and antagonists delivered intracerebrally or intraventricularly on social, stress, and anxiety-related behaviors, while conversely, animal models with genetic disruptions in OT signaling systems exhibit a range of social and behavioral deficits (e.g. see Jin et al., 2007; Young, 2007; Lee et al., 2008; Higashida et al., 2010; Pobbe et al., 2012; Kent et al., 2013; Morales-Rivera et al., 2014; Peters et al., 2014; Burkett et al., 2016; Caldwell et al., 2017; Lee et al., 2018). Collectively, these types of data implicate the central OT signaling system as a promising therapeutic target for a variety of conditions impacting mental health. However, effective therapeutic delivery of exogenous OTR agonists into the CNS of humans remains difficult (Ermisch et al., 1985; McEwen, 2004; Veening and Olivier, 2013; Leng and Ludwig, 2016; Quintana and Woolley, 2016; De Cagna et al., 2019), and thus a more detailed mechanistic understanding of how the brain naturally regulates release of endogenous OT may facilitate development of new approaches for therapeutic manipulation of central OT signaling.

The question of exactly how, and from where, endogenous OT is released to act on both hypothalamic and extrahypothalamic OTRs in the CNS is a complex one. It is complicated by the fact that there are both magnocellular and parvocellular hypothalamic OT synthesizing neurons (OT-MCNs, OT-PCNs), and by the fact that OT-MCNs can release peptide both from their axon terminals and from their dendrites. The current study focuses on dendritic physiology of PVN OT-MCNs because (1) OT-MCNs substantially outnumber OT-PCNs (Althammer and Grinevich, 2017), (2) most axons of OT-MCNs project through the median eminence to the posterior pituitary where activity (action potential) dependent release into the vasculature increases peripheral (and not central) OT concentration (Guzek, 1987; Robinson et al., 1989; Falke, 1991), (3) a large portion of OT available for release into the CNS exists in dendritic rather than axonal vesicles that are subject to activity, and calcium, dependent exocytosis (Pow and Morris, 1989; Ludwig et al., 2002; Ludwig and Leng, 2006), (4) such exocytosis supports functionally important peptide mediated paracrine signaling within the hypothalamus (Son et al., 2013; Smith et al., 2015; Pati et al., 2020), and (5) likely also drives volume transmission to increase functional activation of OTRs in a variety of extrahypothalamic cortical and limbic areas (Veening et al., 2010; Fuxe et al., 2012; Ludwig and Stern, 2015; Brown et al., 2020). Indeed, this mechanism seems likely to work in concert with limited/targeted release from centrally projecting axon collaterals of OT neurons, as has been effectively demonstrated in several extrahypothalamic areas to date (Knobloch et al., 2012; Eliava et al., 2016; Oettl et al., 2016).

In the current study, we use a combination of electrophysiological and subcellular optical recording techniques to evaluate dendritic physiology of OT-MCNs. Somatic activity was induced with a reproducible train of action potential-like voltage pulses delivered to the soma, and calcium influx induced by this somatic activity was quantified using high frequency two-photon line scans across the proximal and distal dendrite. The results reveal that the dendrites of OT-MCNs are weak conductors of somatic voltage changes. We further report that acute hyperosmotic challenge preferentially reduces activity-induced calcium influx in distal vs. proximal OT-MCN dendrites, while acute hypoosmotic challenge increases it. Extensive control experiments indicate these effects are likely mediated by modulation of a previously unidentified osmosensitive channel expressed along the dendritic membrane. Finally, we report that that activity-induced calcium influx in the distal dendrites of OT-MCNs is also preferentially and robustly inhibited, absent any change in osmolarity, by activation of dendritic GABA_A receptors. Collectively, these results significantly increase our understanding of the mechanisms through which OT-MCNs are likely to dynamically regulate the relationship between somatic activity and calcium-dependent dendritic release of OT into the CNS.

This study uses a combination of electrical and optical recording techniques to examine activity-induced calcium influx in the dendrites of PVN OT-MCNs in OT-tdTomato reporter mice. Somatic activity was induced with a well-controlled train of action potential like stimuli delivered to the soma, while activity-induced calcium influx was measured in OT-MCN dendrites using quantitative two-photon microscopy. We demonstrate that PVN OT-MCN dendrites are weak conductors of somatic voltage changes in basal conditions in both male and virgin female mice, and importantly, we also find that activity-induced calcium influx in the dendrites is subject to robust modulation by a diverse set of stimuli acting on distinct types of ion channels in the dendritic membrane.

The primary stimulus used in the current study is an acute increase in osmotic pressure. Hyperosmotic stimuli are well-recognized for an ability to increase activity of hypothalamic MCNs, and in so doing, for promoting significant increases in both peripheral and central OT concentration (Mason, 1980; Bourque and Renaud, 1984; Ludwig et al., 1994; Ludwig, 1998; Tasker et al., 2020). Increases in peripheral OT concentration are understood to depend on axonal release in the neurohypophysis (which delivers OT directly into the vasculature), while increases in central concentration are likely to depend heavily on dendritic release into the extracellular space, the subarachnoid space, and/or the third ventricle (Bourque, 1991; Ludwig and Leng, 2006; Veening et al., 2010). A curious feature of the neurohormonal response to hyperosmotic challenge is that increases in peripheral OT concentration are both rapid and frequency dependent (with concentration closely tracking MCN activity), while increases in central OT concentration are temporally separated from peak MCN firing, often by over an hour (Ludwig et al., 1994; Ludwig, 1998). This is somewhat counter intuitive because like axon terminals, OT-MCN dendrites also contain many large dense core vesicles loaded with oxytocin, and these dendritic vesicles are also subject to both activity and calcium-dependent release (Mason et al., 1986; Pow and Morris, 1989; Ludwig et al., 1995; Wang et al., 1995). Notably, other types of peripheral stimuli (e.g. suckling) can drive more concurrent increases in peripheral and central OT concentration suggesting more synchronous release of OT from both axon terminals and dendrites (Neumann et al., 1993), while some locally synthesized signaling molecules (e.g. α-melanocyte stimulating hormone) have been demonstrated to promote dendritic release of OT while actively inhibiting both firing rate and release from axon terminals in the neurohypophysis (Neumann et al., 1993; Sabatier et al., 2003; Sabatier, 2006). Collectively, these types of data effectively highlight that although at least loosely coupled, the relationship between somatic activity and dendritic release of peptide in hypothalamic MCNs is likely subject to modulation.

The most well-established basis for understanding this flexibility invokes a model of conditional priming, whereby specific endogenous modulators, acting directly on the dendrites, can prime dendritic vesicles to be more available for activity-induced release (Morris and Ludwig, 2004; Ludwig and Leng, 2006). However, in the specific case of hyperosmotic challenge, maximizing calcium-dependent dendritic priming prior to hyperosmotic challenge was found to increase the amount of dendritic release of OT, remarkably, without altering its time course (Ludwig et al., 2002). This striking result suggests that there must be some aspect of OT-MCN physiology that is activated by hyperosmotic challenge, that is separate from conditional priming, and that is capable of rapidly yet transiently reducing the probability of activity-induced exocytosis of dendritic OT.

In that regard, a key finding of the current study is that an acute hyperosmotic stimulus delivered in vitro decreases activity-induced calcium influx in OT-MCN dendrites, while an acute hypoosmotic stimulus increases it. In each case, we noted minimal effect of changing bath conditions on somatic input resistance, or on somatic current observed during the stimulus, and further noted that changes in osmolarity had a more robust effect in distal vs. proximal dendrites. We noted no similar inhibitory effect of acute hyperosmotic stimuli on activity-induced calcium influx in the distal axons of OT-MCNs, indicating compartment specificity, or in the distal dendrites of OT-PCNs or CA1 pyramidal cells, demonstrating cell-type specificity. Further, in experiments that involved both somatic and dendritic stimulation techniques, we demonstrated that hyperosmotic challenge selectively inhibits responses measured distal from the stimulus, irrespective of whether those measurements are made using electrical or optical techniques. Collectively, these data indicate for the first time that osmolarity modulates activity-induced calcium influx in OT-MCN dendrites by acting on osmosensitive ion channels expressed along the dendritic membrane. Based on these data, it is reasonable to postulate that this mechanism may reduce activity-induced dendritic release of OT during times of high somatic activity as induced by acute hyperosmotic challenge. As such, it may be interesting for future studies to evaluate whether low levels of dendritic calcium influx produced by somatic activity when dendritic osmosensitive channels are open, or concurrent action of other dendritic modulators, helps promote priming of dendritic vesicles in a way that contributes to enhanced dendritic release once basal osmolarity is restored.

Another aspect of this study worth specifically highlighting is the novel implication that OT-MCNs express molecularly distinct osmosensitive channels in their soma vs. dendrites. This conclusion is supported by the observation that a non-selective TRP ion channel receptor antagonist, RR, blocked the effect of acute hyperosmotic challenge on action potential firing frequency without altering effects on activity-induced calcium influx as observed in either the proximal or distal dendrites, or the effects on evoked EPSCs produced by a minimal stimulator placed near the distal dendrite. RR is an antagonist of TRPM6, TRPA1, TRPC3, and of all members of the TRPV family (TRPV1-6, Clapham, 2007; Lichtenegger and Groschner, 2014). That said, to our knowledge TRPV channels are the only RR sensitive channels that have potential for osmosensitivity and that are strongly expressed in the hypothalamus (Sharif Naeini et al., 2006; Sladek and Johnson, 2013; Prager-Khoutorsky and Bourque, 2015; Zaelzer et al., 2015; Shenton and Pyner, 2018), suggesting that the somatic osmosensor described here may be a member of the TRPV family. Additional potential effects of RR, for example on ryanodine receptors or mitochondrial calcium transporters, are not expected to be a factor in current experiments since RR is membrane impermeant (Luft, 1971a; Luft, 1971b; Garcha and Hughes, 1994), and we only applied it extracellularly. Additional evidence for the hypothesis that OT-MCNs have distinct somatic and dendritic osmosensors comes from the observation that intracellular cesium blocked the effects of acute hyperosmotic challenge on activity-induced calcium influx in the distal dendrites even through TRPV receptors are cesium permeant (Caterina et al., 1997; Puopolo et al., 2013). Thus, overall, we hypothesize that the somatic osmosensor described here is a member of the TRPV family, while the dendritic osmosensor is, or is coupled to, a cesium sensitive and potassium permeant channel. While some prior literature is generally consistent with these hypotheses (Liu et al., 2005; Sharif Naeini et al., 2006; Zhang et al., 2009; Prager-Khoutorsky and Bourque, 2015), very few studies have examined these questions with respect to OT-MCNs in particular.

Next, it is interesting to highlight that all novel aspects of OT-MCN dendritic physiology revealed here, as well as most other core intrinsic features of OT-MCNs observed, were identical in male and virgin female mice, suggesting that they have a fundamental and sex independent role in regulation of oxytocinergic signaling. That said, it seems plausible that aspects of OT-MCN physiology likely relevant to central OT signaling, such as the dendritic conductivity, could be regulated in a context and sex specific way. As such, it may be interesting to determine whether activity-induced calcium influx in the distal dendrites of OT-MCNs is altered in pregnant or lactating females. Indeed, concurrent increases in both peripheral and central release of OT have been reported in response to suckling (Neumann et al., 1993).

Other aspects of this study make two additional important points. First, we demonstrate directly that the effects of acute hyperosmotic challenge on OT-MCN dendrites are not limited to modulation of activity-induced calcium influx, but also robustly inhibit the somatic response to endogenous synaptic inputs arriving at the distal dendrites. This finding, in combination with other observed effects, suggests that activation of dendritic osmosensors not only transiently reduces the probability of activity-induced dendritic release of OT into the CNS, but also simultaneously reduces the impact of descending central inputs forming dendritic synapses on MCN firing rate. These changes are expected to effectively but transiently prioritize activity-induced increases in peripheral OT concentration. Second, we report that an ability to modulate activity-induced calcium influx by acting on dendritic ion channels can also be produced by stimuli that do not alter osmolarity. Specifically, we note that bath application of a GABA_A receptor agonist not only decreases somatic membrane resistance in OT-MCNs but also preferentially inhibits activity-induced calcium influx in the distal vs. proximal dendrites. The later finding is consistent with prior reports that multiple types of GABAergic receptor subunits, including the typically extrasynaptic δ subunit, are expressed on MCN dendrites and/or in magnocellular regions of the PVN (Fenelon et al., 1995; Pirker et al., 2000; Belelli et al., 2009), and is interesting to consider along with the fact tonic GABAergic currents have been directly observed in MCNs (Park et al., 2006). It is also important in the context of this study because it highlights that endogenous regulation of dendritic conductivity may represent an important mechanism for regulating central OT concentration even in situations where osmolarity remains constant. For example, it is intriguing to note that OT-MCNs receive significantly more frequent bursts of GABAergic IPSCs during lactation (Popescu et al., 2019), and that lactation has also been associated with a significant positive shift in the reversal potential of GABA-receptor-mediated currents (Lee et al., 2015). Overall, we expect that it will be important for future studies to evaluate the ability of additional endogenous modulators to impact conductivity of OT-MCN dendrites, and to identify specific contexts in which such modulators are active.

Finally, as noted in more detail in the Introduction, central OT signaling systems are a promising therapeutic target for a variety of conditions impacting mental health, and yet the best available current strategy for therapeutically modulating activation of central OTRs in humans involves intranasal delivery of an exogenous agonist that has low permeability to the blood brain barrier (Evans et al., 2014; Leng and Ludwig, 2016; Quintana and Woolley, 2016; Quintana et al., 2018). In our view, a better understanding of OT-MCN physiology, particularly as it relates to release of endogenous OT in the CNS, may ultimately lead to improved therapeutic options that are better able to mimic natural concentration, kinetic, and context-dependent aspects of central OT signaling. In the current study, measures of activity-induced calcium influx in OT-MCN dendrites reveals a novel aspect of dendritic physiology likely to underlie the flexible relationship between somatic activity and dendritic release of OT. Future studies may develop new methods for spatially precise quantification of dendritic exocytosis and/or for detection of quantal amounts of OT release very close to the dendritic membrane, which would in turn promote a more direct evaluation of the relationship between dendritic calcium influx and dendritic exocytosis.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures.

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Acceptance summary:

This study examined osmolarity-dependent dendritic signaling in oxytocin magnocellular neurosecretory cells (OT-MNCs). The authors show that repetitive depolarizations evoke larger calcium responses in proximal dendrites relative to distal dendrites. When these neurons were exposed to hyperosmotic stimuli, the distal calcium responses were found to be inhibited to a greater extent compared to proximal dendritic calcium responses. Propagation of glutamate evoked depolarizations from the dendrite towards the soma were also found to be reduced following increases in osmolarity. These effects of hyperosmotic stimuli are likely mediated by changes in membrane resistance of dendrites. A non-selective blocker of the channels, ruthenium red, blocked these effects of hyperosmolarity, indicating the non-selective cation channels (e.g. TRPV types) may be responsible. The finding is significant and addresses fundamental questions about MNC dendritic physiology.

Decision letter after peer review:

Thank you for submitting your article "Dendritic membrane resistance modulates activity-induced Ca²⁺ influx in oxytocinergic magnocellular neurons of mouse PVN" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and John Huguenard as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jeffrey Tasker (Reviewer #1); Rio Teruyama (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

This study examined osmolarity-dependent dendritic signaling in oxytocin magnocellular neurosecretory cells (OT-MNCs). The authors show that repetitive depolarizations evoke larger calcium responses in proximal dendrites relative to distal dendrites. When these neurons were exposed to hyperosmotic stimuli, the distal calcium responses were found to be inhibited to a greater extent compared to proximal dendritic calcium responses. Propagation of glutamate evoked depolarizations from the dendrite towards the soma were also found to be reduced following increases in osmolarity. These effects of hyperosmotic stimuli are likely mediated by changes in membrane resistance of dendrites. A non-selective blocker of the channels, ruthenium red, blocked these effects of hyperosmolarity, indicating the non-selective cation channels (e.g. TRPV types) may be responsible.

All three reviewers agreed that the finding is potentially important and could address fundamental questions about MNC dendritic physiology. However, the reviewers identified a number of technical concerns, as summarized below. These concerns need to be addressed for further consideration.

Essential revisions:

1) The title and abstract are not exactly reflecting what this study is about. The title of the paper is "Dendritic membrane resistance modulates activity-induced Ca²⁺ influx in oxytocinergic magnocellular neurons of mouse PVN". However, dendritic membrane resistance is never actually measured. As such, a title that does not mention membrane resistance may be more appropriate. Also, the purpose and rationale of this study are not clearly communicated in the abstract and introduction. The implication to the regulation of soma-dendritic release of oxytocin, but not hyperosmotic responses, was mentioned in Introduction, while the entire Results and Discussion sections are about hyperosmotic stress.

2) Figure 3: The reviewers believe that stimulation paradigm is not physiological (neurons voltage-clamped at -70 mV with repetitive voltage steps to +50 mV for 5 ms). It is important to show that action potentials in the current clamp, instead of the +50mV voltage step in the voltage-clamp, can produce similar signals.

3) A major focus of the manuscript is on Ca²⁺ elevations in MNC dendrites. However, the authors have not performed the essential experiments to identify what the Ca²⁺ entry/release pathways are. It is important to show that Ca²⁺ is through voltage-gated Ca²⁺ channels for their main conclusions. In addition, it should also be established whether dendritic propagation is active or passive.

4) It is essential to report the effect of the osmotic stimulus alone on dendritic resting Ca²⁺, as this would affect the interpretation of the Ca²⁺ data.

5) Figure 8: What is the effect of RR on proximal EPSCs? This information is needed to interpret the effect of RR on distal EPSCs. It would be required to also test the effect of RR on the modulation of Ca²⁺ responses in distal dendrites to see their effects on the dendritic conductance.

Statistical handling:

Please provide the statistical methods (t-test, 2-way ANOVA with Hom-Sidak corrections, 2-way repeated-measures ANOVA, etc.) used for each measurement in the text or figure legend (not just in the method section). For repeated measures ANOVA, please indicate how measurements were repeated.

For the statistics of sex differences (Figure 2-1, 4-1 etc), it is required to use 3-way ANOVA to assess variability by cells, animals, and sex. The number of males and females used is not clear in some cases, but it appears that only 2 females and 2 males are used (Line 203-204). If this is the case, the statistical comparisons between males and females are not meaningful and should be removed.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Osmosensitive dendritic ion channels modulate activity-induced calcium influx in oxytocinergic magnocellular neurons of the mouse PVN" for further consideration by eLife. Your revised article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and John Huguenard as the Senior Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Essential revisions:

1. This study found were the effect of +15 mOsm ACSF and RR on Ca++ influx in the proximal and distal dendrites. We agree with the authors that the osmosensitive ion channels are most likely responsible for the changes in membrane resistance of dendrites; however, this study did not directly test the ion channels.

We suggest removing "ion channels" from the title, like "Osmolarity regulates activity-induced calcium influx in oxytocinergic magnocellular neurons of the mouse PVN".

2. If the hyperosmotic stimulation is modulating dendritic membrane resistance by opening channels, it would be expected to suppress the dendritic Ca response evoked by activation of the dendrites with glutamate application, if the glutamate-induced Ca response is caused by depolarization. The relative lack of effect of hyperosmotic stimulation on the dendritic Ca response to dendritic glutamate application suggests that the effect is not a simple modulation of membrane resistance.

In the last section of the Results (GABA_A receptor activation), since the voltage change was generated in the soma, it is not surprising that the distal dendritic response would be more attenuated than the proximal dendritic response with GABA_A receptors held open along the length of the dendrite. This doesn't appear to add anything new to the study, since the general increase in membrane conductance caused by tonic GAABAa receptor activation is expected to shunt the response and the shunting effect should be more pronounced further away from the stimulus. It seems that the main take-home message of the study is that dendrites express osmosensitive channels that attenuate the dendritic response and that are different from the osmosensitive channels expressed in the soma.

We suggest discussing this point.

3. As pointed out previously, it is surprising that the proximal dendritic response is not affected by the hyperosmotic stimulation, since the somatic response to current injection (i.e., spiking) is affected, ostensibly via TRPV modulation. Was there a change in the input resistance in the somatic recording in response to the hyperosmotic stimulation that underlies the change in spiking?

Please provide the data of input resistance, and discuss the point mentioned above.

4. In the Results section on distinct somatic vs. dendritic effects of osmotic stimulation (note: osmotic "stress" is still used throughout the manuscript: please fix this), it is implied that a novel finding of the study is that the osmosensor in the soma is a TRP channel. While this is discussed in the Discussion, no mention is made in the Results of the fact that Bourque et al. demonstrated previously that TRPV1 channels are responsible for the osmosensitivity of VP neurons, such that the current findings suggest that the same mechanism is at play in the somatic osmosensitivity of OT neurons. Presumably, the previous findings informed the experiment here to test for TRP channel involvement.

We suggest adding this point in the Result section to clarify the rationale of the experiment.

5. Relatedly, when introducing and interpreting the ruthenium red experiment in the Results, the Bourque findings of a TRPV dependence of the somatic osmosensitivity should be cited. This justifies your testing of RR to target TRP channels and eliminates the implication on lines 338-39 that the somatic sensor being a TRPV channel is a novel finding (although it is new to the OT neurons). Please clarify this point.

Essential revisions:

1) The title and abstract are not exactly reflecting what this study is about. The title of the paper is "Dendritic membrane resistance modulates activity-induced Ca²⁺ influx in oxytocinergic magnocellular neurons of mouse PVN". However, dendritic membrane resistance is never actually measured. As such, a title that does not mention membrane resistance may be more appropriate. Also, the purpose and rationale of this study are not clearly communicated in the abstract and introduction. The implication to the regulation of soma-dendritic release of oxytocin, but not hyperosmotic responses, was mentioned in Introduction, while the entire Results and Discussion sections are about hyperosmotic stress.

In response to this comment, we have changed the title to: “Osmosensitive dendritic ion channels modulate activity‐induced calcium influx in oxytocinergic magnocellular neurons of the mouse PVN”. This change both removes the term ‘dendritic membrane resistance’ from the title and draws more attention to the focus on osmosensitive mechanisms. That said, please note that experiments presented in Figure 9 demonstrate that activity‐induced calcium influx in OT‐MCN dendrites can also be similarly modulated by stimuli that do not change osmotic pressure. We believe that this is an important point in the overall scope of the manuscript. As such, text in the abstract has been updated to explicitly reference effects of both osmosensitive and non‐osmosensitive ion channels on activity‐induced calcium influx in OT‐MCN dendrites, and both types of results are further previewed in the Introduction.

2) Figure 3: The reviewers believe that stimulation paradigm is not physiological (neurons voltage-clamped at -70 mV with repetitive voltage steps to +50 mV for 5 ms). It is important to show that action potentials in the current clamp, instead of the +50mV voltage step in the voltage-clamp, can produce similar signals.

In response to this comment, we have completed a new experiment in current clamp that evaluates the effect of acute hyperosmotic challenge on activity‐induced calcium influx, in both proximal and distal dendrites of OT‐MCNs, using a train of 250 μs long suprathreshold current pulses, still delivered at 20 Hz for 2 sec, to evoke action potentials. The amplitude and kinetics of action potentials produced by this stimulus closely matched spontaneous action potentials observed in the same cells. Using this protocol, we found that acute hyperosmotic challenge reduced activity‐induced calcium influx in the proximal and distal dendrites by ‐13.3 ± 3.61% and ‐38.3 ± 3.92%, respectively. These results closely matched those observed with the original protocol (reduction of ‐14.3 ± 3.37% and ‐40.6 ± 4.23% in the proximal vs. distal dendrites, respectively). These new data are now presented in the Results section of the manuscript, and in more detail in Figure 4-figure supplement 3.

3) A major focus of the manuscript is on Ca²⁺ elevations in MNC dendrites. However, the authors have not performed the essential experiments to identify what the Ca²⁺ entry/release pathways are. It is important to show that Ca²⁺ is through voltage-gated Ca²⁺ channels for their main conclusions. In addition, it should also be established whether dendritic propagation is active or passive.

In response to the first point, we performed a new experiment that evaluates the effect of removing extracellular calcium on activity‐induced calcium influx, as observed in both proximal and distal dendrites of OT‐MCNs. Specifically, we found that transient (15‐minute) bath application of calcium free ACSF reduces activity‐induced calcium influx observed in the proximal and distal dendrites by 80.3 ± 3.5% and by 67.9 ± 3.3%, respectively, and that the activity‐induced calcium signal rapidly recovers when extracellular calcium is restored. These results confirm that influx of extracellular calcium is the primary driver of activity‐induced calcium influx measured in our experiments. Given that the stimulus is somatic depolarization of the patched neuron only, and that both AMPA and NMDA receptors are blocked, these results further support the conclusion that VGCCs are required, without eliminating a possible downstream contribution of calcium induced calcium release. These data are now presented in the Results section and are illustrated in Figure 3-figure supplement 1. Additional data generated with low calcium and high magnesium ACSF (0.25 mM and 10 mM, respectively, intended to directly block a subset of VGCCs) also strongly reduced activity‐induced calcium influx in proximal dendrites (by 56.9 ± 2.3%, n=2).

With respect to the second point, about whether dendritic propagation is active or passive, we expect it is primarily passive because there is substantial loss of signal with increasing distance from the soma in control conditions, which is not observed in cells filled with a cesium based internal, as presented in Figure 3. That said, to more directly evaluate the possibility of a minor role for voltage gated sodium channels in supporting voltage propagation in OT‐MCN dendrites, we have now performed an additional analysis of the data from TTX experiments presented in Figure 4. Specifically, we calculated the ratio of the activity-induced calcium signal in distal vs. proximal dendrites of OT‐MCNs both before and after bath application of TTX. The results indicate that TTX has no impact on this ratio (n=5, ratio = 0.52 ± 0.1 and 0.40 ± 0.1 before and after bath application of TTX, respectively, t=1.43, p=0.23, two‐sample paired t‐test). Based on this analysis, which has been added to the Results section of the manuscript, we can now more confidently conclude that voltage‐gated sodium channels do not appear to contribute meaningfully to voltage propagation in OT‐MCN dendrites.

4) It is essential to report the effect of the osmotic stimulus alone on dendritic resting Ca²⁺, as this would affect the interpretation of the Ca²⁺ data.

In response to this comment, we have now completed a more thorough analysis of the effect of acute hyperosmotic challenge on resting calcium, in both proximal and distal dendrites, throughout the manuscript. We find that on average, basal calcium levels (as indicated by resting F/F) changed by ≤ 6.1% in response to acute hyperosmotic challenge, and importantly, that small fluctuations in resting calcium were not correlated with observed effects of acute hyperosmotic challenge on activity‐induced calcium influx. This conclusion is now directly stated in the text of the results and is presented in greater detail in Figure 4-figure supplement 2.

5) Figure 8: What is the effect of RR on proximal EPSCs? This information is needed to interpret the effect of RR on distal EPSCs. It would be required to also test the effect of RR on the modulation of Ca²⁺ responses in distal dendrites to see their effects on the dendritic conductance.

In response to the later parts of this comment, we have completed a new experiment which reveals that pre‐treatment with RR does not alter the effect of acute hyperosmotic challenge on activity‐induced calcium influx, as observed in either the proximal or distal dendrites of OT‐MCNs. The results have been added to Figure 8, in place of the results of experiments with distally evoked EPSCs. The eEPSC experiment is still presented in Figure 8-figure supplement 1. Collectively, we now provide data indicating that pre-treatment with RR does not change the effect of acute hyperosmotic challenge on activity‐induced calcium influx in either proximal or distal dendrites, or the effect of acute hyperosmotic challenge on the amplitude of distally evoked EPSCs. We did not evaluate the ability of RR to modulate the effect of acute hyperosmotic challenge on proximally evoked EPSCs in part because we believe this question is better addressed by the new calcium imaging data now highlighted in Figure 8, and in part because even absent RR, the effect of acute hyperosmotic challenge on proximally evoked EPSCs is significantly smaller than that observed using distally evoked EPSCs, as reported in Figure 7.

Statistical handling:

Please provide the statistical methods (t-test, 2-way ANOVA with Hom-Sidak corrections, 2-way repeated-measures ANOVA, etc.) used for each measurement in the text or figure legend (not just in the method section). For repeated measures ANOVA, please indicate how measurements were repeated.

We now provide statistical methods used in line with each result in either the main text or figure legend, and we have significantly updated the methods section covering statistical analysis to more clearly indicate when regular vs. repeated measures ANOVAs were used. Factors for each analysis are now also more specifically highlighted, and when present, factors with repeated measures are also noted.

For the statistics of sex differences (Figure 2-1, 4-1 etc), it is required to use 3-way ANOVA to assess variability by cells, animals, and sex. The number of males and females used is not clear in some cases, but it appears that only 2 females and 2 males are used (Line 203-204). If this is the case, the statistical comparisons between males and females are not meaningful and should be removed.

In response to this comment, we have removed the two‐sample proportion test previously used to evaluate co‐localization of tdTomato and NP1 in PVN neurons of males vs. females. Although other datasets used to evaluate sex differences have more animals available (as noted in text and/or source data files), we do not believe animal is a particularly informative/viable factor for a 3‐way ANOVA because in many cases only a small number of cells could be recorded per animal. As such, we use two‐way ANOVA with sex and cell type or dendritic location as factors for these analyses in the manuscript. That said, in response to this comment we did run a 3‐way ANOVA using data presented in Figure 1‐1 and 4‐1, with sex, animal, and cell‐type or dendritic location as factors, and noted that populations means separated by animal were not significantly different in any dataset, and that there were no significant interactions between animal and any other factor.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential revisions:

1. This study found were the effect of +15 mOsm ACSF and RR on Ca++ influx in the proximal and distal dendrites. We agree with the authors that the osmosensitive ion channels are most likely responsible for the changes in membrane resistance of dendrites; however, this study did not directly test the ion channels.

We suggest removing "ion channels" from the title, like "Osmolarity regulates activity-induced calcium influx in oxytocinergic magnocellular neurons of the mouse PVN".

In response to this comment, we have changed the title to: “Dendritic osmosensors regulate activity-induced calcium influx in oxytocinergic magnocellular neurons of the mouse PVN.” This change removes the term “ion channels”, as requested, but retains an important key word ‘dendritic’.

2. If the hyperosmotic stimulation is modulating dendritic membrane resistance by opening channels, it would be expected to suppress the dendritic Ca response evoked by activation of the dendrites with glutamate application, if the glutamate-induced Ca response is caused by depolarization. The relative lack of effect of hyperosmotic stimulation on the dendritic Ca response to dendritic glutamate application suggests that the effect is not a simple modulation of membrane resistance.

Figure 5 highlights that whether the stimulus is somatic current injection, or dendritic depolarization produced by local application of glutamate, inhibition by acute hyperosmotic challenge is only apparent / robust when the response is measured distal from the stimulus that produced it. These results effectively highlight the effect of acute hyperosmotic challenge on dendritic conductivity.

By contrast, the relative lack of effect of hyperosmotic stimulation on the dendritic Ca response to dendritic glutamate application (measured at the site of application) does not mean that there is no change in local dendritic membrane resistance at all. Rather, it indicates that depolarization produced by local application of glutamate still drives the local membrane potential to a voltage that is suprathreshold for opening of voltage gated calcium channels. Importantly, we expect that any effect of hyperosmotic challenge on dendritic Rm occurs not just at the site of glutamate application, but along the whole length of the dendrite. Thus, we expect even a small local change in dendritic Rm (that is subthreshold for altering local glutamate evoked calcium influx) can have an additive effect on signal propagation, leading to a clear effect on glutamate‐evoked current arriving at the soma.

In the last section of the Results (GABA_A receptor activation), since the voltage change was generated in the soma, it is not surprising that the distal dendritic response would be more attenuated than the proximal dendritic response with GABA_A receptors held open along the length of the dendrite. This doesn't appear to add anything new to the study, since the general increase in membrane conductance caused by tonic GAABAa receptor activation is expected to shunt the response and the shunting effect should be more pronounced further away from the stimulus. It seems that the main take-home message of the study is that dendrites express osmosensitive channels that attenuate the dendritic response and that are different from the osmosensitive channels expressed in the soma.

We suggest discussing this point.

If GABA_A receptors were purely somatic, then the ratio of the effect in the distal dendrite vs. the proximal dendrite would be expected to be the same before and after muscimol, even though there would be less activity‐induced calcium influx at the distal dendrite than at the proximal dendrite in both conditions. In contrast, we find that muscimol, like acute hyperosmotic challenge, reduces activity-induced calcium influx at the distal dendrites more strongly than it does at the proximal dendrites. In our opinion, this is an important result in part because it highlights a role for GABA_A receptors expressed along the length of the dendrite in modulating activity‐induced calcium influx in OT‐MCNs. To our knowledge, this has not been functionally demonstrated in hypothalamus before, or more importantly, in neurons capable of releasing peptide from their dendrites in a calcium‐ and activity‐dependent manner. That said, this result is also quite important in our view because it indicates that dynamic regulation of dendritic conductance is likely to underlie the relationship between action potential firing and dendritic release of peptide from OT‐MCNs all the time, and not just in conditions that involve changes in osmolarity. Indeed, we expect further studies may reveal a range of additional endogenously available signaling molecules that will act on dendrites to modulate activity‐induced calcium influx at sites of dendritic peptide release. It is our hope that learning more about the range of signaling molecules and effector systems that can impact this relationship will ultimately help inform new therapeutic strategies for manipulating endogenous oxytocin signaling in the CNS.

We agree that a main take‐home message of the study is that dendrites express osmosensitive channels that attenuate the dendritic response and that are different from the osmosensitive channels expressed in the soma. These points are covered directly in the discussion (see sections beginning on line 407 and 426).

3. As pointed out previously, it is surprising that the proximal dendritic response is not affected by the hyperosmotic stimulation, since the somatic response to current injection (i.e., spiking) is affected, ostensibly via TRPV modulation. Was there a change in the input resistance in the somatic recording in response to the hyperosmotic stimulation that underlies the change in spiking?

Please provide the data of input resistance, and discuss the point mentioned above.

In response to the first part of this comment, it is important to note that activity‐induced calcium influx as observed at the proximal dendrite is significantly reduced by acute hyperosmotic challenge (Δ Peak ΔF/F proximal: ‐14 ± 3.37%, n = 13, t = ‐4.2 p – 0.001, as reported on line 178). This result was also reproduced, although in revision 1 not explicitly highlighted as statistically significant, in Figure 4 —figure supplement 3, using a current clamp protocol (Δ Peak ΔF/F proximal: ‐13.3 ± 3.6% n = 7, t = ‐3.7, p = 0.01). In our view, these results are likely produced primarily by activation of dendritic osmosensors along the first ~25 μm of the dendrite, and the effect that has on signal propagation between the soma and the site of dendritic calcium imaging. We have updated the text to better highlight these observations (see in particular updated legends for Figure 4 and Figure 4 —figure supplement 3).

In response to the second part of this comment, we acknowledge that acute hyperosmotic challenge (at +15 mOsm) had no statistically significant effect on somatic membrane resistance as measured using a standard membrane test protocol in voltage clamp, despite having a modest but statistically significant effect on spontaneous action potential firing frequency as observed in current clamp (+1.39 ± 0.55 Hz, as reported in Figure 2C). In response to prior reviews, we noted that it is likely that this change in frequency could be produced by quite a small change in membrane potential. Given the high basal input resistance of OT‐MCNs (well over 1 GΩ on average), we expect the somatic osmosensitive current underlying the effect on spontaneous firing frequency, and thus any associated change in somatic Rm associated with gating of osmosensors, is also quite small. Finally, we noted that although not statistically significant, in raw terms, somatic membrane resistance did decrease in response to acute hyperosmotic challenge (by 78 MΩ), consistent with other data that suggest opening rather than closing of a somatic conductance, such as TRPV1.

In the current revision, to evaluate this issue more thoroughly, we have completed additional voltage clamp experiments where we measured somatic membrane resistance in OT‐MCNs during acute hyperosmotic challenge under identical conditions, and with an identical protocol, but without concurrent 2P imaging of the dendrites, ultimately increasing n value in this dataset from 21 to 38. The additional data had no impact on the average somatic membrane resistance measured either before or after acute hyperosmotic challenge (before: 1435 ± 187.5 MΩ vs. 1355 ± 109.8 MΩ, n=21, 38, t = 0.40, p = 0.69, unpaired t‐test, after: 1,357 ± 177.9 MΩ vs. 1256 ± 109.3 MΩ, n=21, 38, t = 0.52, p = 0.61, unpaired t‐test) and the updated dataset continued to indicate that acute hyperosmotic challenge has no statistically significant effect on somatic membrane resistance (before hyperosmotic challenge: 1355 ± 109.8 MΩ, after: 1256 ± 109.3 MΩ, n=38, t = 1.38, p=0.18, paired t‐test). In the original dataset, mean somatic membrane resistance as observed in response to acute hyperosmotic challenge decreased by 78 MΩ (5.4%), and in the updated dataset it decreased by 99 MΩ (7.3%). A power analysis conducted using population means and overall variance from the updated dataset (n=38) indicates that 159 replicates would be necessary to detect an effect of this magnitude with an α level of 0.05 and power of 0.80. Importantly, this analysis does not mean that a very small change in somatic membrane resistance is not occurring, it simply means that any effect that does exist is too small to observe with statistical significance given our available sample size and overall population variance. This core result has been updated in the manuscript on line 182.

In response to the final part of this comment, we are not able to quantify the effect of acute hyperosmotic challenge on somatic membrane resistance in the same cells where the effect on spontaneous firing frequency was evaluated because those cells were recorded in current clamp during the osmotic stimulus using a protocol designed to continuously monitor spontaneous firing frequency.

4. In the Results section on distinct somatic vs. dendritic effects of osmotic stimulation (note: osmotic "stress" is still used throughout the manuscript: please fix this), it is implied that a novel finding of the study is that the osmosensor in the soma is a TRP channel. While this is discussed in the Discussion, no mention is made in the Results of the fact that Bourque et al. demonstrated previously that TRPV1 channels are responsible for the osmosensitivity of VP neurons, such that the current findings suggest that the same mechanism is at play in the somatic osmosensitivity of OT neurons. Presumably, the previous findings informed the experiment here to test for TRP channel involvement.

We suggest adding this point in the Result section to clarify the rationale of the experiment.

We have modified this section of the results to more directly describe prior work implicating TRPV1 channels in osmosensation of vasopressinergic neurons.

All remaining references to osmotic ‘stress’ have been removed from this section, and throughout the manuscript.

5. Relatedly, when introducing and interpreting the ruthenium red experiment in the Results, the Bourque findings of a TRPV dependence of the somatic osmosensitivity should be cited. This justifies your testing of RR to target TRP channels and eliminates the implication on lines 338-39 that the somatic sensor being a TRPV channel is a novel finding (although it is new to the OT neurons). Please clarify this point.

Edits made in response to point 4 above also now more explicitly indicate that prior work by Bourque and colleagues on VP neurons informed the choice of RR for present experiments.

Author details

1. Wanhui Sheng

   Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9357-0771

2. Scott W Harden

   Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, United States

   Contribution

   Conceptualization, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0757-1979

3. Yalun Tan

   1. Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, United States
   2. Department of Anesthesiology, School of Medicine, Stanford University, Stanford, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Eric G Krause

   1. Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, United States
   2. Center for Integrative Cardiovascular and Metabolic Diseases, University of Florida, Gainesville, United States
   3. Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville, United States

   Contribution

   Resources, Supervision, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2718-3113

5. Charles J Frazier

   1. Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, United States
   2. Center for Integrative Cardiovascular and Metabolic Diseases, University of Florida, Gainesville, United States

   Contribution

   Conceptualization, Resources, Software, Formal analysis, Supervision, Funding acquisition, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   cjfraz@ufl.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3550-4789

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