Volume 93, Issue 1, Pages 57-65 (January 2017) Bidirectional Anticipation of Future Osmotic Challenges by Vasopressin Neurons  Yael Mandelblat-Cerf, Angela Kim, Christian R. Burgess, Siva Subramanian, Bakhos A. Tannous, Bradford B. Lowell, Mark L. Andermann  Neuron  Volume 93, Issue 1, Pages 57-65 (January 2017) DOI: 10.1016/j.neuron.2016.11.021 Copyright © 2017 Terms and Conditions

Figure 1 Water Cues and Drinking Both Cause Rapid Drops in Spiking Activity of Individual VPpp Neurons (A) Selective expression of ChR2-mCherry in SON vasopressin neurons that project to the posterior pituitary (VPpp). (B and C) Spiking activity for two example VPpp neurons. Both neurons demonstrated a rapid decrease in firing within seconds of presentation of the lickspout (green vertical dashed line), and again at onset of water availability (black vertical dashed line). Vertical ticks indicate licking prior to (yellow) and during (orange) water consumption. Significant decreases in firing from a baseline period prior to lickspout placement were observed in the 1 min period following lickspout placement, and in the 1 min period following access to water. Example spike waveforms (averaged across 10 spikes) confirm that endogenous spike waveform shapes (black) remained stable across the recording and match the shape of photostimulation-evoked spike waveforms (blue). (D) Time course of percent change in firing from pre-lickspout baseline (green vertical line) for all identified VPpp neurons recorded during this task (n = 14). Short vertical black lines denote the onset of water availability. Example neurons in (B) and (C) are labeled “#1” and “#2.” The dashed gray line indicates the end of the period of manual lickspout positioning and adjustment, a period excluded from subsequent analyses. (E) Percent change in spiking rate of VPpp neurons and unidentified SON neurons for different drinking periods. Error bars denote SEM. Analyses included n = 10 VPpp neurons and n = 27 unidentified SON neurons for the pre-drinking period (see Experimental Procedures). For all other periods, all VPpp neurons (n = 14) and unidentified neurons (n = 34) were included. Paired t tests: ∗p < 0.05, ∗∗p < 0.01. See also Figures S1–S3. Neuron 2017 93, 57-65DOI: (10.1016/j.neuron.2016.11.021) Copyright © 2017 Terms and Conditions

Figure 2 Water Cues and Drinking Both Cause Rapid Drops in VPpp Population Activity across Multiple Hyperosmolar Contexts (A) Upper panel: GCaMP6 fiber photometry measurements of population activity in VPpp neurons via a 200 μm fiber in SON. Lower panel: example VPpp population activity after 24 hr of water deprivation, demonstrating a fast drop within seconds of presentation of a water bowl (green vertical line) and an additional drop at drinking onset (gray vertical line). Inset: zoom-in of activity surrounding bowl placement and drinking onset. (B) Change in VPpp neuron activity across drinking periods, demonstrating a significant drop before drinking onset (n = 5 mice). (C) In contrast to water-restricted mice (blue trace), sated mice (gray trace, n = 3) with free access to water showed no decrease in VPpp neuron activity following water bowl presentation (p > 0.15 for periods from 0–30 s and 30–60 s). (D) As with water restriction, water cues and drinking following consumption of dry food both caused a rapid drop in VPpp population activity (n = 5 mice). (E) In contrast to these sustained responses to drinking (light blue trace), unexpected presentation of an empty bowl lacking water (gray trace) caused a transient drop in VPpp neuron activity (at both 5–10 and 10–15 s after placement; ∗paired t test, p < 0.05, Bonferroni corrected for multiple comparisons) that promptly returned to baseline levels. Paired t tests in (B) and (D): ∗p < 0.05, ∗∗p < 0.01. Bonferroni corrected for multiple comparisons. Error bars denote SEM. See also Figure S3. Neuron 2017 93, 57-65DOI: (10.1016/j.neuron.2016.11.021) Copyright © 2017 Terms and Conditions

Figure 3 Feeding Onset and Offset Also Induce Rapid VPpp Population Responses (A–C) VPpp population response to ad libitum ingestion of dry food from a food bowl (A), and to presentation of 250 mg food pellets (B), 15 mg pellets (C, black) and intermingled presentation of non-food items of similar size (C, red). Upper traces: examples. Lower traces: average responses across mice. Shaded error bars denote SEM (n = 5 mice). Beige shaded regions denote periods of food availability. (D–F) Average VPpp population responses to feeding. Error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, paired t test with Bonferroni correction for multiple comparisons. Lower asterisks indicate significant differences from baseline activity. Upper asterisks indicate significant differences from activity in the period prior to feeding offset. n.s.: not significant. See also Figure S3. Neuron 2017 93, 57-65DOI: (10.1016/j.neuron.2016.11.021) Copyright © 2017 Terms and Conditions

Figure 4 Highly Reliable Pre-ingestive VPpp Population Responses to Water Cues but Not to Food Cues (A–E) Single-trial time courses of increases (red), decreases (blue), or no change (white) in VPpp population activity relative to the period prior to food item placement (A–C) or water bowl placement (D and E). Trials are sorted according to latency from item placement to estimated ingestion onset (vertical black ticks). Left column is aligned to item presentation (green vertical line); right column is aligned to ingestion onset. (A–C) Ingestion of food from a bowl (A), a large food pellet (B), or a small food pellet (C) following food restriction. (D and E) Ingestion of water following 24 hr water deprivation (D) or following consumption of dry food (E). Activity increased within 20 s of consumption for 100% (12/12) of food bowl events, 99% (116/117) of large pellet events, and for 93% (135/145) of small pellet events. In contrast, activity decreased in 96% (51/53) of feeding-induced drinking events and 100% (12/12) of water deprivation-induced drinking events. We then considered changes in activity following item presentation but prior to ingestion (for all events in which this pre-consumption window lasted at least 5 s). The water cue evoked a decrease in activity in 89% (32/36) and 91% (10/11) of trials that followed chow feeding and water deprivation, respectively. In contrast, presentation of food cues did not evoke consistent increases in activity in food-restricted mice prior to ingestion onset (51%, 35/68; 51%, 27/53; and 33%, 4/12). (F1) Similarly, average cue-evoked changes in activity across trials revealed a significant drop following water bowl presentation but prior to drinking (p < 0.01), but no response to presentation of food items prior to feeding (all p values > 0.1). Error bars denote SEM. (F2) In contrast, significant changes from pre-cue baseline to the 10 s period following onset of ingestion were observed for both feeding and drinking (∗∗p < 0.01). Error bars denote SEM. (G) Schematic illustrating the various presystemic inputs that are likely to contribute to the fast increases and decreases in VPpp neuron activity during ingestive behavior. “X” indicates that pre-ingestive food cue signals to do not appear to affect VPpp neuron activity. Neuron 2017 93, 57-65DOI: (10.1016/j.neuron.2016.11.021) Copyright © 2017 Terms and Conditions