Dendritic membrane resistance modulates activity-induced Ca2+ influx in oxytocinergic magnocellular neurons of mouse PVN

Hypothalamic oxytocinergic magnocellular neurons have a fascinating ability to release peptide from both their axon terminals and from their dendrites. Existing data indicates there is a flexible relationship between somatic activity and dendritic release, but the mechanisms governing this relationship are not completely understood. Here we use a combination of electrical and optical recording techniques to quantify activity-dependent 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, and yet activity-induced dendritic calcium influx can be robustly regulated by a diverse set of stimuli that open or close ionophores located along the dendritic membrane. Overall, this study reveals that dendritic membrane resistance is a dynamic and endogenously regulated feature of OT-MCNs that is likely to have substantial functional impact on central oxytocin release. IMPACT STATEMENT Activity-induced dendritic calcium influx in oxytocinergic magnocellular neurons can be robustly modulated by a highly diverse set of stimuli acting on distinct types of ionophores expressed along the dendritic membrane.

dependent release into the vasculature increases peripheral (and not central) OT concentration 47 (Guzek, 1987;Robinson et al., 1989;Falke, 1991), 3) a large portion of OT available for release into 48 the CNS clearly exists in dendritic rather than axonal vesicles that are subject to activity, and calcium, 49 dependent exocytosis ( Indeed, this mechanism seems likely to work in concert with limited/targeted release from centrally 55 projecting axon collaterals of OT neurons, as has been effectively demonstrated in several 56 extrahypothalamic areas to date (Knobloch et al., 2012;Eliava et al., 2016;Oettl et al., 2016). 57 In the current study we use a combination of electrophysiological and subcellular optical recording 58 techniques to evaluate dendritic physiology of OT-MCNs. Somatic activity was simulated with a 59 reproducible train of action potential-like voltage pulses delivered to the soma, and calcium influx 60 induced by this somatic activity was quantified using high frequency two-photon line scans across the 61 proximal and distal dendrite. The results reveal that the dendrites of OT-MCNs are weak conductors 62 of somatic voltage changes. We further report that hyperosmotic stress preferentially reduces activity-63 dependent calcium influx in distal vs. proximal OT-MCN dendrites, while hypoosmotic stress 64 increases it. Extensive control experiments indicate these effects are likely mediated by modulation 65 of a previously unidentified osmosensitive channel expressed along the dendritic membrane. Finally, 66 we report that that activity-induced calcium influx in the distal dendrites of OT-MCNs is also 67 preferentially and robustly inhibited, absent any change in osmotic stress, by activation of dendritic 68 GABAA receptors. Collectively, these results significantly increase our understanding of the 69 mechanisms through which the brain is likely to dynamically regulate the relationship between 70 somatic activity and calcium-dependent dendritic release of OT into the CNS. 71

72
Animals 73 All experiments in this study were performed using 1-3-month-old OT-reporter mice which expresses 74 red fluorescent protein (tdTomato) in oxytocinergic neurons. These mice were generated by crossing 75 OT-IRES-Cre knock-in mice ( internal solution). All cells were permitted to rest for 15 minutes following establishment of whole-167 cell configuration to ensure robust diffusion of fluorophore into dendrites before proceeding with 168 calcium imaging experiments. Somatic activity was induced in cells voltage-clamped at -70 mV using 169 a train of brief action potential like voltage steps (to +50 mV for 5 ms, delivered at 20 Hz for 2 seconds). 170 Activity-dependent calcium influx was measured in dendrites or axons using two-photon line scans 171 across specific structures of interest (11.9 s duration, 85 Hz) at known distances from the soma. 172 Emissions from Fluo-5F (green, G) and Alexa Fluor 594 (red, R) were separated with a dichroic mirror 173 and simultaneously measured by two separate photomultiplier tubes (

196
All experiments for this study were performed in an OT-reporter mouse line designed to selectively 197 expresses a red fluorescent protein (tdTomato) in oxytocinergic neurons ( Figure 1A, see Methods). In 198 order to validate specificity and selectivity of this reporter in the PVN, we used immunohistochemical 199 techniques to evaluate co-expression of tdTomato and neurophysin 1 (NP1, an OT carrier protein 200 found in oxytocinergic neurons, Figure 1B). Across all animals tested (n=2 male, 2 female), we found 201 that over 92% of tdTomato-expressing neurons in PVN were immunoreactive for NP1 (2629/2838). 202 Importantly, we also noted that 1262/1352 and 1367/1468 of tdTomato positive neurons examined 203 were NP1 positive in males and females, respectively, indicating that there is no sex based difference 204 in the extent of co-localization (Z=1.38, p=0.17). Based on these data, we used a combination of IR-205 DIC and epifluorescence microscopy (see Fig. 1C and

221
Significant prior evidence suggests that OT-MCNs are likely to be osmosensitive, and further suggest 222 that osmosensitivity may reveal mechanisms that allow OT-MCNs to modulate the relationship 223 between cellular activity and release of OT into the CNS. In order to directly evaluate the 224 osmosensitivity of PVN OT-MCNs in vitro, we used whole-cell current clamp recordings, in 225 combination with bath application of mannitol (MT, an inert osmolyte), to evaluate the effect of acute 226 hyperosmotic stress on firing frequency. We found that bath application of 30 mOsm MT for 5 227 minutes (in the presence of glutamate and GABA receptor antagonists, see Methods) increased basal 228 firing rate of OT-MCNs by 3.37 ± 0.55 Hz (n = 9, t=6.2, p = 0.0002, Fig. 2A observed an overall increase in activity-induced calcium influx (F2.7, 23.9=9.45, p= 3.9 x 10 -4 vs. 257 observations in K-glu, Fig. 3C, red trace), with post-hoc tests indicating increased response at 258 distances > 60 µm from the soma). These results clearly indicate that functional calcium indictor is 259 present in the distal dendrites, and that voltage-gated calcium channels are still robustly expressed at 260 distal locations. As such, they also substantially reinforce the conclusion that loss of activity-261 dependent calcium influx at increasing distance from the soma, as observed when using a more 262 physiological K-gluconate based internal solution, was produced by loss of current through open 263 channels along the dendritic membrane. 264 Next, we repeated the experiment for a third time, using a K-glu internal, to evaluate activity-265 dependent calcium influx along the length of OT-MCN axons. Axons were distinguished from 266 dendrites based primarily on their smaller initial diameter as observed with 2P epifluorescence 267 microscopy (e.g. Fig. 4A). However, consistent with prior reports (Hatton, 1990;Stern and Armstrong, 268 1998), we also noted that axons often (but not always) branch off a primary dendrite very close to the 269 soma. The results of this experiment indicated that OT-MCN axons, unlike dendrites, reliably and 270 actively propagate somatic voltage changes in cells filled with potassium gluconate (Fig. 3C,  Acute osmotic stress preferentially reduces activity-dependent calcium influx in 277 distal vs. proximal dendrites of OT-MCNs. 278 We next tested the hypothesis that acute hyperosmotic stress (as produced by bath application of 15 279 mOsm MT) directly modulates the relationship between somatic activity and activity-dependent 280 dendritic calcium influx in OT-MCNs. Towards that end, we used a technical approach very similar to 281 that employed in Fig. 3, however instead of measuring activity-induced influx at multiple locations 282 under control conditions, we picked just two dendritic locations (proximal and distal to the soma) and 283 repeatedly measured activity-induced calcium influx before and after acute hyperosmotic stimulation. 284 Proximal dendritic locations were located within 25 µm of the soma, while distal ones were located at 285 ~125 µm from the soma (See Fig. 4A, blue and red dashed lines, respectively). In order to generate 286 activity-induced calcium influx at these locations the soma was stimulated with the same 2-sec 20 Hz 287 train of action potential like voltage steps as used in Fig. 3, 2P line scan data were collected from each 288 dendritic location and analyzed in an identical manner, and experiments were again performed in the 289 continuous presence of bath applied antagonists for glutamate and GABA receptors (see previous 290 Results section and Methods). We found that acute hyperosmotic stimulation had a mild inhibitory 291 effect on activity-induced calcium influx in the proximal dendrites of OT-MCNs (-12.6 ± 5.15 %, n = 292 11, t = -2.4, p = 0.034), and yet strikingly, had a much stronger inhibitory effect in the distal dendrites 293 (-40.8 ± 4.0%, n = 21, t = 4.2, p = 0.0002 vs. proximal, Fig. 4B, Fig. 4C Fig. 4D). Collectively, these data clearly indicate that despite having 311 an excitatory effect on action potential frequency as observed in the soma (Fig. 2), acute hyperosmotic 312 stress also preferentially reduces activity-dependent calcium influx as observed in the distal vs. 313 proximal dendrites of OT-MCNs, strongly suggesting a change in dendritic membrane resistance. 314 Effects of acute osmotic stress on activity-dependent calcium influx in distal 315 dendrites are very likely mediated by changes in dendritic membrane 316 resistance.

317
To further strengthen the conclusion that the results in Fig. 4 are produced by an osmosenstive change 318 in dendritic membrane resistance, we performed an additional control experiment designed to further 319 rule out alternative mechanisms. Specifically, we initiated whole-cell patch clamp recordings from 320 OT-MCNs using the same K-glu internal as in Fig. 4, but now without glutamate receptor antagonists 321 in the bath. We then stimulated them alternately (every 15 seconds) with two distinct stimuli (one 322 delivered to the soma and one to the distal dendrites). The somatic stimulus was the same 2 sec 20 323 Hz train of action potential like voltage steps used in Fig. 3-4, while the dendritic stimulus was focal 324 application of exogenous glutamate (accomplished using a picospritzer, See Methods). For each 325 stimulus, responses were measured in both the soma (as a whole-cell current) and in a distal dendrite 326 (as ΔG/R generated with a 2P line scan as in Figs. 3-4). Collectively, this experimental design (Fig. 5A) 327 provides insight, in each individual OT-MCN tested, on how acute hyperosmotic stress effects current 328 propagating along the dendrite in both directions, either from the soma towards the distal dendrites, 329 or from the distal dendrites towards the soma. The results emphasize that acute hyperosmotic stress 330 consistently and selectively inhibits whichever response is measured distal from the stimulus that 331 produced it, irrespective of whether that response is measured using electrical or optical techniques. 332 For example, hyperosmotic stress reduced somatic current observed in response to exogenous 333 glutamate delivered to the distal dendrite by 62.5 ± 10.1% (n = 5, t = -6.17, p = 0.003, Fig. 5B), and 334 yet had no effect on somatic current observed in response to a train of AP like voltage pulses delivered 335 to the soma (ΔCurrent = -2.4 ± 4.53%, n = 10, t = -0.5, p = 0.612, Fig. 5B). Conversely, hyperosmotic 336 stress reduced dendritic calcium influx produced by delivering a train of voltage pulses to the soma 337 (by -33.13 ± 7.5 %, n = 10, t = -4.4, p = 0.002, Fig. 5C), and yet had no effect on dendritic calcium 338 influx observed in response to locally applied glutamate (Δ Peak Δ F/F: 1.94 ± 8.7 % of baseline, n = 339 10, t = 0.22, p = 0.828, Fig. 5C). Overall, we believe that these results, in combination with other data 340 presented above, effectively rule out the idea that the observed effects of acute hyperosmotic stress 341 depend on direct modulation of voltage-gated calcium channels, calcium induced calcium release, or 342 on other aspects of calcium homeostasis in the dendrites. As such, they further strengthen the 343 conclusion that acute osmotic stress is directly modulating dendritic membrane resistance ( Changes in dendritic membrane resistance produced by acute hyperosmotic 357 stress are cell type specific, compartment specific, and bidirectional. 358 Next, to eliminate any possible generalized or nonspecific effects of acute hyperosmotic stress, we 359 designed experiments to test the hypothesis that the effects are both compartment specific and cell 360 type specific. Compartment specificity was evaluated using techniques identical to those employed for 361 Fig. 4, except that we compared activity-dependent calcium influx in the distal dendrite to that 362 observed in the distal axon. We found that acute hyperosmotic stress again effectively inhibited 363 activity-dependent calcium influx in distal dendrites (Δ Peak Δ F/F : -44.0 ± 8.0 %, n = 5, t = -5.5, p 364 = 0.005), and yet produced a much smaller effect in the distal axon (Δ Peak Δ F/F :-10.0 ± 2.3 %, n = 365 5, t = 3.4, p = 0.027 vs. distal dendrite, Fig. 6A). This result demonstrates significant compartment 366 specificity within individual OT-MCNs. In order to evaluate cell type specificity, we used identical 367 approaches to measure activity-dependent calcium influx in proximal vs. distal dendrites in PVN OT 368 PCNs (identified as described in Fig. 1), and in CA1 pyramidal cells. In OT PCNs there was no 369 significant effect of acute hyperosmotic stress in proximal or distal dendrites (Δ Peak Δ F/F : -7.7 ± 370 4.3 %, n = 5, t = -1.8, p = 0.147; 6.6 ± 3.4 %, n = 5, t=1.96, p=0.12, respectively, Fig. 6B). In CA1 371 pyramidal cells, we noted a mild inhibitory effect in proximal dendrites, but no effect in distal 372 dendrites (-6.4 ± 2.4 %, t = -2.7, p = 0.045; -3.80 ± 3.5 %, n = 6, t = -1.1, p = 0.328, respectively, Fig.  373 6C). These results effectively demonstrate cell type specificity. Next we reasoned that if acute 374 hyperosmotic stress inhibits calcium induced influx in the distal dendrites of OT-MCNs by opening a 375 distinct dendritic osmosensitive ion channel, and if that channel is not completely closed in control 376 conditions, then an acute hypoosmotic stimulus should have opposite effects. Indeed, we found that 377 acute reduction of osmolarity by 30 mOsm (achieved by diluting the bath solution with water) 378 effectively increased activity-dependent calcium influx in the distal dendrites of OT-MCNs (by 31 ± 379 10.7 % of baseline, n = 10, t = 3.0, p = 0.014, Fig. 6D), while having no effect in the proximal dendrites 380 (n = 10, t = -0.6, p = 0.536, Fig. 6D). As with effects of hyperosmotic stress, these changes occurred 381 absent any significant effect on somatic membrane resistance or voltage clamp current observed 382 during stimulation (n=10, t=1.3, p=0.21; n=10, t=-1.84, p=0.1, respectively). Collectively, these results 383 demonstrate that the effects of acute osmotic stress on activity-induced calcium influx, as observed in 384 the distal dendrites of OT-MCNs, are compartment specific, cell type specific, and bidirectional. 385 Changes in dendritic membrane resistance as produced by acute osmotic stress 386 preferentially inhibit minimally evoked EPSCs produced by a distal vs. proximal 387 stimulator.

388
Data presented in Fig. 5 indicates that the somatic response to activation of glutamate receptors on 389 the distal dendrites of OT-MCNs is significantly inhibited by acute osmotic stress. This result (in 390 combination with other results above) suggests that changes in dendritic membrane resistance are 391 likely to impact integration of synaptic inputs, as well as activity-dependent calcium influx. In order 392 to test this hypothesis directly we used minimal stimulation techniques (See Methods) to evoke 393 glutamate release from one or few axons that make synaptic contact with either the proximal or distal 394 dendrites of an OT-MCN (Fig. 7A). After identifying a clear evoked excitatory postsynaptic current 395 (eEPSC), we bath applied 15 mM MT as in prior experiments. We found that acute hyperosmotic stress 396 reliably and reversibly reduced eEPSC amplitude as evoked by a minimal stimulator placed near the 397 distal dendrite (by 63.4 ± 8.7%, n = 8, t = -7.3, p = 1.6 x 10 -4 , Fig. 7A,C). As in earlier experiments, this 398 result was not associated with a significant change in somatic membrane resistance (n=8, t=-0.14, 399 p=0.90). If, as expected, the effect is instead produced primarily by a drop in dendritic membrane 400 resistance, then the same acute osmotic stimulus should have less of an inhibitory effect on EPSCs 401 generated using a minimal stimulator placed near the proximal dendrite. Indeed, consistent with this 402 hypothesis, we found that 15 mM MT reduced proximally evoked EPSC amplitude by 30 ± 9.6% (n=6, 403 t=-3.16, p=0.03, Fig. 7A,B). Consistent with our hypothesis, this effect is significantly smaller than 404 observed when using a stimulator placed near the distal dendrite (t=-2.55, p=0.03). In order to 405 confirm that eEPSCs involved in these experiments were glutamatergic, a subset of MT sensitive 406 responses (n=3) were challenged with bath applied glutamate receptor antagonists after recovery, and 407 were effectively eliminated (not illustrated). 408 the soma compared to the dendrites, we hypothesized that somatic and dendritic osmosensors in  MCNs are likely to be molecularly distinct. In order to test this hypothesis directly we evaluated the 418 effect of ruthenium red (RR), a generic antagonist of transient receptor potential cation channels in 419 subfamily V (TRPV receptors), on both somatic and dendritic effects of acute hyperosmotic stress 420 observed in OT-MCNs (as in Figs. 2 and 7, respectively). Consistent with our hypothesis, we found 421 that pre-treatment with 10 µM RR blocked the effect of hyperosmotic stress on basal firing rate as 422 observed in current clamp (Δ action potential frequency: 0.28 ± 0.21, n=7, t=0.5, p=0.61 vs. null 423 hypothesis of mean=0, t=4.0, p=0.001 vs. response to same stimulus absent RR, Fig. 8A-B). 424

Distinct somatic vs. dendritic effects of acute osmotic stress on OT-MCNs are
Conversely, RR did not block the inhibitory effect of acute hyperosmotic stress on evoked EPSCs as 425 observed in a separate group of cells in voltage clamp (Δ EPSC amplitude: -71.5 ± 6.0%, n=6, t=-11.9, 426 p<0.001 vs. null hypothesis of mean=0, t=0.72, p=0.49 vs. effect observed absent RR, Fig. 8C-D). 427 These results reinforce the hypothesis that PVN OT-MCNs express distinct osmosensors in somatic 428 vs. dendritic compartments, and further suggest that the somatic but not dendritic osmosensor may 429 be a member of the TRPV family. Interestingly, we also noted that the effect of MT on activity-induced 430 calcium influx as observed in the distal dendrites of OT-MCNs is blocked in cells filled with a cesium-431 gluconate internal solution, which suggests that the dendritic osmosensor may be cesium sensitive 432 (See Extended Data Fig. 8-1 for additional details). 433 GABAA receptor activation preferentially reduces activity-dependent calcium 434 influx in distal vs. proximal dendrites of OT-MCNs. 435 Next, we sought to determine whether dendritic membrane resistance in OT-MCNs is an aspect of 436 dendritic physiology that can be actively manipulated to modify the relationship between somatic 437 activity and dendritic calcium influx, even under conditions that do not involve changes in osmotic 438 stress. Towards that end, we performed experiments similar to those presented in Fig. 4, except we 439 replaced the hyperosmotic stimulus with bath application of 400 nM muscimol (a GABAA receptor 440 agonist). Unlike hyperosmotic stress, bath application of muscimol significantly reduced somatic 441 membrane resistance (from 1178 ± 166.6 MΩ to 226 ± 43.5 MΩ, n = 6, t = 4.9, p = 0.004) and 442 produced a tonic inhibitory current (of 15.4 ± 5.42 pA, n = 6, t = 2.8, p = 0.036) apparent in cells 443 voltage clamped at -70 mV, consistent with tonic activation of somatic GABAA receptors. However, 444 importantly, like acute hyperosmotic stress, muscimol preferentially inhibited activity-induced 445 calcium influx in the distal vs. proximal dendrites (-75.8 ± 2.7 % vs. -31.6 ± 6.4 %, respectively, n=6, 446 t=5.6, p=0.003, Fig. 9). This finding is consistent with opening of GABA receptors along the dendritic 447 membrane (see also Pirker et al., 2000;Park et al., 2006), and notably, highlights that dendritic 448 membrane resistance is likely to be under constant and dynamic regulation in OT-MCNs even absent 449 changes in osmotic stress. 450

451
This study uses a combination of electrical and optical recording techniques to examine activity-452 dependent calcium influx in the dendrites of PVN OT-MCNs in OT-tdTomato reporter mice. Somatic 453 activity was induced with a well-controlled train of action potential like stimuli delivered to the soma, 454 while activity-dependent calcium influx was measured in OT-MCN dendrites using quantitative 2-455 photon microscopy. We demonstrate that PVN OT-MCN dendrites are weak conductors of somatic 456 voltage changes in basal conditions in both male and virgin female mice, and importantly, we also 457 find that activity-induced calcium influx in the dendrites is subject to robust modulation by a diverse 458 set of stimuli acting on distinct types of ionophores in the dendritic membrane. 459 The primary stimulus used in the current study is an acute increase in osmotic pressure. peak MCN firing, often by over an hour (Ludwig et al., 1994;Ludwig, 1998). This is somewhat counter 470 intuitive because like axon terminals, OT-MCN dendrites also contain many large dense core vesicles 471 loaded with oxytocin, and these dendritic vesicles are also subject to both activity and calcium 472 dependent release (Mason et al., 1986;Pow and Morris, 1989;Ludwig et al., 1995;Wang et al., 1995). 473 Notably, other types of stimuli can drive more synchronous release of OT from both axons and 474 dendrites, or can preferentially promote dendritic release (Neumann et al., 1993b;Sabatier et al., 475 2003). Collectively these types of data effectively highlight that although at least loosely coupled, the 476 relationship between somatic activity and dendritic release of peptide in hypothalamic MCNs is highly 477 flexible. 478 The most well-established basis for understanding this flexibility invokes a model of conditional 479 priming, whereby specific endogenous modulators, acting directly on the dendrites, are able to prime 480 dendritic vesicles to be more available for activity-dependent release (Morris and Ludwig, 2004; 481 Ludwig and Leng, 2006). However, in the specific case of osmotic stress, maximizing calcium 482 dependent dendritic priming prior to hyperosmotic stimulation was found to increase the amount of 483 dendritic release of OT, remarkably, without altering its time course (Ludwig et al., 2002). This 484 striking result suggests that there must be some aspect of OT-MCN physiology that is activated by 485 hyperosmotic stimulation, that is separate from conditional priming, and that is capable of rapidly yet 486 transiently reducing the probability of activity-dependent exocytosis of dendritic OT. 487 In that regard, a key finding of the current study is that an acute hyperosmotic stimulus delivered in 488 vitro decreases activity-induced calcium influx in OT-MCN dendrites, while an acute hypoosmotic 489 stimulus increases it. In each case, we noted minimal effect of changing bath conditions on somatic 490 input resistance, or on somatic current observed during the stimulus, and yet changes in osmotic 491 stress had a more robust effect in distal vs. proximal dendrites. We noted no similar inhibitory effect 492 of acute hyperosmotic stimuli on activity-induced calcium influx in the distal axons of OT-MCNs, 493 indicating compartment specificity, or in the distal dendrites of OT-PCNs or CA1 pyramidal cells, 494 demonstrating cell-type specificity. Further, in experiments that involved both somatic and dendritic 495 stimulation techniques, we demonstrated that hyperosmotic stimulation selectively inhibits 496 responses measured distal from the stimulus, irrespective of whether those measurements are made 497 using electrical or optical techniques. Collectively, these data indicate for the first time that osmotic 498 stress modulates activity-dependent calcium influx in OT-MCN dendrites by acting on osmosensitive 499 ion channels expressed along the dendritic membrane. Based on these data, we believe it is reasonable 500 to postulate that this mechanism may reduce activity-dependent dendritic release of OT during times 501 of high somatic activity as induced by acute hyperosmotic stress. As such, it may be interesting for 502 future studies to evaluate whether low levels of dendritic calcium influx produced by somatic activity 503 when dendritic osmosensitive channels are open, or concurrent action of other dendritic modulators, 504 helps promote priming of dendritic vesicles in a way that contributes to enhanced dendritic release 505 once basal osmolarity is restored. 506 Another aspect of this study worth specifically highlighting is the novel implication that OT-MCNs 507 express molecularly distinct osmosensitive channels in their soma vs. dendrites. This conclusion is 508 supported by the observation that a generic TRPV receptor antagonist blocked the effect of 509 hyperosmotic stress on action potential firing frequency without altering effects on EPSCs produced 510 by a minimal stimulator placed near the distal dendrite. It is further strengthened by the observation 511 that intracellular cesium blocked the effects of hyperosmotic stress on activity-dependent calcium 512 influx in the distal dendrites even through TRPV receptors are cesium permeant ( these questions with respect to OT-MCNs in particular. 518 Next, it is interesting to highlight that all novel aspects of OT-MCN dendritic physiology revealed here, 519 as well as most other core intrinsic features of OT-MCNs observed, were identical in male and virgin 520 female mice, suggesting that they have a fundamental and sex independent role in regulation of 521 oxytocinergic signaling. That said, it seems plausible that aspects of OT-MCN physiology likely 522 relevant to central OT signaling, such as the dendritic membrane resistance, could be regulated in a 523 context and sex specific way. As such, it may be interesting to determine whether activity-dependent 524 calcium influx in the distal dendrites of OT-MCNs is naturally increased in pregnant or lactating 525 females. Indeed, concurrent increases in both peripheral and central release of OT have been reported 526 in response to suckling (Neumann et al., 1993). 527 Other aspects of this study make two additional important points. First, we demonstrate directly that 528 the effects of acute hyperosmotic stimulation on OT-MCN dendrites are not limited to modulation of 529 activity-dependent calcium influx, but also robustly inhibit the somatic response to endogenous 530 synaptic inputs arriving at the distal dendrites. This finding, in combination with other observed 531 effects, suggests that acute systemic osmotic stress not only transiently reduces the probability of 532 activity-dependent dendritic release of OT into the CNS, but also simultaneously reduces the impact 533 of descending central inputs forming dendritic synapses on MCN firing rate. These changes are 534 expected to effectively but transiently prioritize activity-dependent increases in peripheral OT 535 concentration. Second, we report that an ability to modulate activity-dependent calcium influx by 536 acting on dendritic ionophores is not unique to osmotic stress. Specifically, we note that bath 537 application of a GABAA receptor agonist not only decreases somatic membrane resistance in OT-538 MCNs but also preferentially inhibits activity-induced calcium influx in the distal vs. proximal 539 dendrites. The later finding is consistent with prior reports that GABAergic receptors are expressed 540 on MCN dendrites (Pirker et al., 2000;Park et al., 2006), and it is important in the context of this 541 study because it highlights that endogenous regulation of dendritic conductivity may represent an 542 important mechanism for regulating central OT concentration even in situations that have nothing to 543 do with osmotic stress. Therefore, we expect that it will be important for future studies to evaluate the 544 ability of additional endogenous modulators to impact conductivity of OT-MCN dendrites. 545 Finally, as noted in more detail in the Introduction, central OT signaling systems are a promising 546 therapeutic target for a variety of conditions impacting mental health, and yet the best available 547 current strategy for therapeutically modulating activation of central OTRs in humans involves 548 intranasal delivery of an exogenous agonist that has low permeability to the blood brain barrier (Evans  549  et  Core intrinsic properties of OT-MCNs (blue) and OT-PCNs (red) did not differ by sex as illustrated in 588 panels A-D. Although intrinsic properties did not vary by sex within either cell type, 3 of 4 of these 589 properties did differ significantly across cell type. Specifically, in OT-MCNs vs. OT-PCNS, membrane 590 resistance was significantly larger, transient current observed in voltage clamp after a step from -70 591 mV to -50 mV was outward rather than inward, and delay to first spike observed during a 592 suprathreshold stimulation in current clamp was significantly longer. All three of these differences 593 were apparent in both males and females. Key comparisons between cell types, and within sex, are 594 highlighted by brackets in panels A-C (* = p<0.05, while *** = p<0.001). Somewhat surprisingly, we 595 found that whole-cell capacitance was not significantly different in OT-MCNs vs. OT-PCNs, in either 596 sex (panel D). 597 Change in spontaneous action potential frequency over time in response to +30 mOsm (n=9, top) and 602 +15 mOsm (n=9, bottom). C) Both 15 and 30 mOsm stimuli caused a significant increase in action 603 potential frequency in OT-MCNs (denoted by † † †, p<0.001), but response to 30 mOsm stimulus was 604 significantly larger (denoted by * p< 0.01, blue vs. green bar). By contrast, the same 30 mOsm 605 stimulus did not produce a significant increase in firing rate in OT-PCNs (orange bar, p=0.06 vs. null 606 hypothesis of mean=0), and these results were significantly different that those observed in OT-MCNs 607 (** denotes p = 0.003). 608  t=0.56, p=0.6). As such, these data were combined across sex and presented in Fig. 4C, left panel. 656 hyperosmotic stress on somatic responses to either dendritic or somatic stimulation (purple, red, 669 respectively). C) Illustrates the effect of acute hyperosmotic stress on dendritic responses to either 670 dendritic or somatic stimulation (purple, red, respectively). Collectively these results highlight that 671 bath application of 15 mOsm MT effectively and selectively inhibits responses that are measured distal 672 from the stimulus that produced them, irrespective of whether those responses are measured with 673 electrical or optical techniques. These data reinforce the conclusion that acute hyperosmotic stress 674 reduces dendritic membrane resistance. † † p<0.01. 675 osmotic stress selectively and preferentially increases (rather than decreases) activity-dependent 685 calcium influx observed in the distal dendrites of OT-MCNs. As in prior experiments on distal 686 dendrites, all line scans for these experiments were conducted at ~125 µm from the soma, and acute 687 hyperosmotic stress was produced by bath application of 15 mOsm. Acute hypoosmotic stress (of -30 688 mOsm) was produced by diluting ACSF in the bath with water. ** p≤0.01. 689  action potential firing frequency in OT-MCNs is blocked in cells pretreated with RR. C) Raw data 706 from a representative OT-MCN illustrating that acute hyperosmotic stress continues to inhibit 707 minimally evoked EPSCs evoked at the distal dendrite, even in cells pretreated with RR. D) Summary 708 data highlighting that pretreatment with RR has no impact on the ability of acute hyperosmotic stress 709 to inhibit distally evoked EPSCs. Note that control datasets in panels B and D were previously 710 presented in Fig. 2C and Fig. 7D, respectively. ** p=0.001, † † † p≤0.001. 711 somatic activity were measured at proximal and distal sites (dashed lines) before and after bath 726 application of 300 nM muscimol (a GABAA receptor agonist). Other than using muscimol in place of 727 acute hyperosmotic stress, techniques are identical those described for Fig. 4. B) Representative 728 calcium responses observed during somatic stimulation in the proximal (blue) vs. distal (red) dendrite 729 of an OT-MCN, before (lighter trac) and after (darker trace) bath application of muscimol. C) 730 Summary data indicates that bath application of muscimol produces greater inhibition of activity-731 dependent calcium influx in the distal vs. proximal dendrites of OT-MCNs. *** p<0.001. 732