Dendritic osmosensors modulate activity-induced calcium influx in oxytocinergic magnocellular neurons of the mouse PVN
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Abstract
Hypothalamic oxytocinergic magnocellular neurons have a fascinating ability to release peptide from both their axon terminals and from their dendrites. Existing data indicates that the relationship between somatic activity and dendritic release is not constant, but the mechanisms through which this relationship can be modulated are not completely understood. Here, we use a combination of electrical and optical recording techniques to quantify activity-induced calcium influx in proximal vs. distal dendrites of oxytocinergic magnocellular neurons located in the paraventricular nucleus of the hypothalamus (OT-MCNs). Results reveal that the dendrites of OT-MCNs are weak conductors of somatic voltage changes; however, activity-induced dendritic calcium influx can be robustly regulated by both osmosensitive and non-osmosensitive ion channels located along the dendritic membrane. Overall, this study reveals that dendritic conductivity is a dynamic and endogenously regulated feature of OT-MCNs that is likely to have substantial functional impact on central oxytocin release.
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This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on December 14 2020, follows.
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 …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on December 14 2020, follows.
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
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.
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.
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.
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.
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 (Fig. 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.
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