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Thirst-associated preoptic neurons encode an aversive motivational drive

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Science  15 Sep 2017:
Vol. 357, Issue 6356, pp. 1149-1155
DOI: 10.1126/science.aan6747
  • Fig. 1 TRAP2 efficiently and specifically labels dehydration-activated neurons in the median preoptic nucleus (MnPO).

    (A and B) TRAP2 design and principle. (C) Experimental timelines to determine the efficiency and specificity of Thirst-TRAP, by comparing Thirst- or Homecage-TRAP tdTomato expression with Fos immunolabeling in response to 48 hours of water deprivation (Thirst-Fos) or 4 hours at 37°C (Warm-Fos). (D) tdTomato expression in MnPO after recombination of TRAP2;Ai14 mice, under water-satiated (Homecage-TRAP) and 48-hours water-deprived (Thirst-TRAP) conditions. a.c., anterior commissure; D, dorsal; V, ventral; M, medial; L, lateral. (E) Representative confocal images of tdTomato/Fos overlap for three conditions, as indicated above. (F) Quantification of total tdTomato induction in MnPO after recombination in Homecage- or Thirst-TRAP mice; t test. (G) Efficiency and specificity of MnPO Thirst-TRAP. (H) TRAP/Fos overlap (Double+/Fos+) for the three experimental groups, one-way ANOVA, Holm-Šidák correction. Numbers of mice quantified for each experiment are in parentheses. **P < 0.01, ****P < 1 × 10−4. Data are presented as mean ± SEM.

  • Fig. 2 Molecular identity of MnPO Thirst-TRAPed neurons.

    (A) Single-cell RNA-sequencing of MnPO Thirst-TRAPed neurons. Tissue containing MnPO of Thirst-TRAP mice is microdissected from live brain slices. Neurons are dissociated then FAC-sorted into 96-well plates. Single-cell cDNA libraries are prepared and sequenced together. (B) t-distributed stochastic neighbor embedding (t-SNE) representation of 348 transcriptomes showing two clusters. (C) Top 30 differentially expressed genes between the two clusters, per cluster, sorted by average difference in expression per cluster and Z-scored per gene. (D and E) (Top) t-SNE representation of cells colored by Gad1 (D) or Slc17a6 (E) expression. (Bottom) Expression of Gad1 or Slc17a6 in cells within Cluster 1 (purple) and Cluster 2 (green). Expression, log-normalized TPM (transcripts per million) values (see methods). (F) Three-color smFISH of tdTomato (red), Gad1 (blue), and Slc17a6 (green) in the MnPO (dotted oval) of a Thirst-TRAP mouse. (Left) Low-magnification view; two fields of view highlighted. (Right) High-magnification view of tdTomato+ (red) cells outside MnPO (1) or within MnPO (2), along with Gad1 and Slc17a6 expression in the same cells, indicated with arrows. (G) Fraction of tdTomato+Slc17a6+ and tdTomato+Gad1+ neurons within MnPO, out of total tdTomato+ cells. N = 2 mice. tdT, tdTomato. (H to J) Double smFISH of tdTomato and Cluster 2 markers, Adcyap1, Agtr1a, or Nxph4, in Thirst-TRAP mice. (Top left) Low-magnification view of marker and tdTomato with MnPO circled. (Top right) High-magnification view of tdTomato (red) and marker gene (green) expression. Arrows, double-labeled cells. (Bottom left) t-SNE representation of cells colored by expression of marker gene. (Bottom right) Fractional combinations of tdTomato+ with each marker gene. Adcyap1: N = 276 total cells; Agtr1a: 396 total cells; Nxph4: 402 total cells, across N = 3 mice per marker gene.

  • Fig. 3 Optogenetic activation of MnPO Thirst-TRAPed neurons induces a scalable, aversive thirst motivational state.

    (A) Experimental timeline for Thirst- and Homecage-TRAP to express a virally delivered effector. (B) (Left) Fiber-optic implant for illuminating MnPO. (Right) Viral constructs for Cre-inducible iC++ or ChR2 expression. (C) Total licks in water-restricted mice expressing iC++ in MnPO during 7.5-min laser-ON or laser-OFF session; paired t test. (D) Licking for water after 20-Hz stimulation (blue) of Thirst-TRAPed MnPO neurons expressing ChR2 in 5 water-satiated mice. (E and F) Total licks over 15-min prestimulation or stimulation period of N = 5 Thirst- (E) or Homecage- (F) TRAPed MnPO neurons at 2.5, 5, 10, and 20 Hz. (G) Cumulative reinforcements (water rewards) over 20 min, before, during (blue), and after 20-Hz stimulation, after training. N = 5 animals per group. (H and I) Total reinforcements within 15 min prestimulation or stimulation period of N = 5 water-satiated Thirst- (H) or Homecage- (I) TRAPed MnPO neurons at 2.5, 5, 10, and 20 Hz. (J) Real-time place preference (RTPP) to 20-Hz stimulation. (K) Aggregate RTPP data of difference in time on nonstimulated and stimulated sides; unpaired t test. (L) Reinforcements per 30-min session of mice learning to lever-press to receive a 20-s break in otherwise constant 20-Hz stimulation. Two-way ANOVA, Holm-Šidák correction. (M) Cumulative reinforcements over 30-min session. N = 5 Homecage- (gray) and N = 5 Thirst- (green) TRAP mice. (N) Total reinforcements within a 30-min session for Thirst-TRAP mice lever-pressing to turn off 0-, 5-, 10-, and 20-Hz MnPO stimulation; Kruskal-Wallis test, false discovery rate correction. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant. Data presented as mean ± SEM.

  • Fig. 4 MnPO thirst-associated neurons integrate water intake to control motivational drive.

    (A) Diagram of possible models relating MnPO activity dynamics and behavior. Behavioral trace represents idealized cumulative reinforcements as a mouse lever-presses for water. (B) Diagram of MnPO fiber photometry recordings with multiplexed Ca2+-independent control (405 nm) and Ca2+-dependent (490 nm) illumination. (C) Coexpression of GCaMP6s and tdTomato in Thirst-TRAP MnPO. (D) Single-trial MnPO activity dynamics upon drinking water in a water-restricted Thirst-TRAP mouse. (E) (Top) Average MnPO activity upon licking full (green) or empty (black) water bottle. (Bottom) Lick rate over time. N = 5 mice. (F) Single-trial MnPO activity dynamics during FR1 lever-pressing in water-restricted Thirst-TRAP mouse. (G) (Top) Average MnPO activity during task performance on a FR1 (green) or Extinction (black) schedule. (Extinction, no water delivered.) (Middle) Average licking behavior. (Bottom) Average cumulative reinforcements. N = 5 mice, averaged across N = 3 sessions per mouse. (H) Same as (G) but for mouse on FR6 schedule. (I) Quantification of average fluorescence during 5-min interval from T = 10 min to T = 15 min, after presentation of empty bottle or water bottle (E), or after onset of Extinction and FR1 schedule lever-pressing for water (G); paired t test. (J and K) Lick rate (J) or Z-scored MnPO activity (K) in 2-min bins for free drinking from water bottle (E), FR1 lever-pressing (G), and FR6 lever-pressing (H). (L) Time for activity to decrease to half minimal value for each mouse, based on sigmoid fit; two-way ANOVA, Benjamini-Hochberg correction. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant. Data presented as mean ± SEM.

Supplementary Materials

  • Thirst-associated preotic neurons encode an aversive motivational drive

    William E. Allen, Laura A. DeNardo, Michael Z. Chen, Cindy D. Liu, Kyle M. Loh, Lief E. Fenno, Charu Ramakrishnan, Karl Deisseroth, Liqun Luo

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S9
    • Table S1
    • References

    Images, Video, and Other Media

    Movie S1
    Optogenetic stimulation of MnPO neurons rapidly evokes drinking in water-sated mice The mouse quickly approaches the water cap and begins drinking when blue laser light (5 ms pulses at 20 Hz) is delivered, and the mouse stops drinking after the laser is turned off. Optogenetically-induced drinking was quantified at different stimulation frequencies (Fig. 3E). Note that the mouse does not exhibit undirected gnawing or licking during stimulation.

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