1. Neuroscience

A limbic circuit selectively links active escape to food suppression

  1. Estefania P Azevedo
  2. Bowen Tan
  3. Lisa E Pomeranz
  4. Violet Ivan
  5. Robert Fetcho
  6. Marc Schneeberger
  7. Katherine R Doerig
  8. Conor Liston
  9. Jeffrey M Friedman  Is a corresponding author
  10. Sarah A Stern  Is a corresponding author
  1. Laboratory of Molecular Genetics, The Rockefeller University, United States
  2. Department of Psychiatry, Weill Cornell Medical College-New York Presbyterian Hospital, United States
  3. Howard Hughes Medical Institute, The Rockefeller University, United States
https://doi.org/10.7554/eLife.58894
Share this article
Cite this article
  1. Estefania P Azevedo
  2. Bowen Tan
  3. Lisa E Pomeranz
  4. Violet Ivan
  5. Robert Fetcho
  6. Marc Schneeberger
  7. Katherine R Doerig
  8. Conor Liston
  9. Jeffrey M Friedman
  10. Sarah A Stern
(2020)
A limbic circuit selectively links active escape to food suppression
eLife 9:e58894.
https://doi.org/10.7554/eLife.58894
  2. Download BibTeX
  3. Download .RIS
5 figures, 1 video, 1 table and 4 additional files

Figures

Figure 1 with 1 supplement
Download asset Open asset
Identification of lateral septum neurons activated by acute restraint stress.

(A) Activity mapping of the whole brain using c-fos staining in control (naïve) and restraint-stressed (RS) mice. Areas with the highest c-fos expression (green) difference are shown: the basolateral amygdala (BLA), bed nucleus of the stria terminals (BNST), insular cortex (IC), and lateral septum (LS). Insets show a 2× digital zoom of c-fos expression. Scale bars, 50 μm. (B) Activity-based transcriptomics (PhosphoTrap) of the lateral septum of control (naïve) and restraint-stressed mice. Middle panel, plot depicting the average IP/INP value (log2) of all genes analyzed in control and restraint stress (RS) samples. Enriched genes (>2-fold; red) and depleted genes (<2-fold; blue) are shown, and activation or target gene markers are shown: Nts (neurotensin), Fos, and osb (n = 2). All genes depicted have q-values <0.05 as calculated by Cufflinks. (C) mRNA levels of Nts evaluated using Taqman qPCR in control and restraint stress (RS) samples: control (black bars) and RS (red bars) are compared (n = 6). Unpaired Student’s t-test, *p<0.05, t = 2.692, df = 10. Data are represented as mean ± SEM. (D) In situ hybridization showing the expression of Nts (green) and Fos (red) mRNA in males exposed to acute restraint stress (RS) or naïve (control). Quantification of the number of Nts+/Fos+ cells per slice/mice are shown, n = 6, Unpaired Student’s t-test, *p<0.05, t = 2.962, df = 10. Scale bar, 25 μm. Data are represented as mean ± SEM.

Figure 1—figure supplement 1
Download asset Open asset
LSNTS neurons are activated by acute restraint stress.

(A) Representative images of c-fos (green) in the lateral septum of control and restraint stress (RS) mice. Right, quantification of c-fos+ cells in control (black) and RS (red) mice. n = 4, Unpaired Student’s t-test, *p<0.05, t = 3.964, df = 4. Scale bars, 50 um. (B) Representative images of in situ hybridization experiments measuring the levels of Fos (red) and Nts (green) in the lateral septum of control and restraint stress (RS) male mice. Left, quantification of Fos+ cells in control (black) and RS (red) male mice, n = 4, Nested t-test, ****p<0.0001, t = 5.175, df = 34. Right, quantification of Nts+ cells in control (black) and RS (red) male mice, n = 4, Nested t-test, **p<0.01, t = 3.476, df = 34. Scale bars, 50 um.

Figure 2 with 2 supplements
Download asset Open asset
LSNts neurons regulate food intake and body weight in mice.

(A) Left panel, experimental scheme. Right panel, food intake, measured in grams (g), in control (black) and RS (red) mice after 60 and 120 min following RS. n = 5(RS), n = 6(control), Two-way ANOVA with post-hoc Bonferroni correction, Time: F(1,9)=175.9, p<0.0001; Subject: F(1,9)=16.86, p=0.003; Interaction: F(1,9)=4.975, p=0.0527. (B) Left panel, experimental scheme. Nts-cre mice were injected with an AAV encoding the hM3Dq activatory DREADD receptor in the LS. Middle panel, cumulative food intake, measured in grams (g) of control mCherry (black) and hM3Dq (red) expressing Nts-cre mice after 60 and 120 min following CNO injection. n = 7(mCherry), n = 10(hM3Dq), Two-way ANOVA with post-hoc Bonferroni correction, Time: F(1,15)=55.46, p<0.0001; Subject: F(1,15)=12.84, p=0.0027; Interaction: F(1,15)=5.45, p=0.034. (C) Daily food intake, measured in grams (g) daily during injections of saline (days 1–3, 7–8) or CNO (days 4–6) in control mCherry (black) or hM3Dq (red) expressing Nts-cre mice. n = 8; Two-way ANOVA with post-hoc Bonferroni correction, Time: F(7,72)=2.871, p=0.01; Subject: F(1,72)=46.92, p<0.0001; Interaction: F(7,72)=6.48, p<0.0001. (D) Body weight (delta), measured in grams (g) after 3 d of daily CNO injections in control mCherry (black) or hM3Dq (red) expressing Nts-cre mice. n = 8; Unpaired Student’s t-test, *p<0.05, t = 2.903, df = 10. (E) Left panel, experimental scheme. Right panel, cumulative food intake, measured in grams (g), in control naïve mice (control, black) or mice that underwent restraint stress (RS, red), measured 2 hr after re-feeding after an overnight fast, n = 6. Unpaired Student's t-test: T(10)=2.427, p=0.0356. (F) Left panel, experimental scheme. Right panel, cumulative food intake, measured in grams (g), in control mCherry (black) or hM3Dq (red) expressing Nts-cre mice, measured 2 hr following re-feeding after an overnight fast, n = 8, Paired Student’s t-test, *p<0.05, t = 3.171, df = 7. (G) Left panel, experimental scheme. Right panel, cumulative food intake, measured in grams (g), in control mCherry (black) and hM4Di (red) expressing Nts-cre mice after 60 and 120 min following saline injection. n = 7(mCherry), n = 8(hM4Di). Two-way ANOVA with post-hoc Bonferroni correction, Time: F(1,19)=415.8, p<0.0001; Subject: F(1,13)=1.042, p=0.326; Interaction: F(4,52)=2.839, p=0.0333. (H) Daily food intake, measured in grams (g) after daily injections of saline (days 1–3, 7–8) or CNO (days 4–6) in control mCherry (black) or hM4Di (red) expressing Nts-cre mice. n = 7; Two-way ANOVA with post-hoc Bonferroni correction, Time: F(7,96)=3.949, p=0.0008; Subject: F(1,96)=18.95, p<0.0001; Interaction: F(7,96)=0.876, p=0.5285. (I) Body weight (delta), measured in grams (g) after 3 d of daily CNO injections in control mCherry (black) or hM4Di (red) expressing Nts-cre mice. n = 8; Unpaired Student’s t-test, p=0.83, t = 0.2168, df = 10. Data are represented as mean ± SEM.

Figure 2—figure supplement 1
Download asset Open asset
LSNts neurons regulate feeding.

(A) Representative images of viral injection strategy and viral expression (mCherry, red) and c-fos (green) in control virus (left) and hM3Dq (right) injected mice following CNO injection. Right, quantification of c-fos+ cells in mCherry control (black) and hM3Dq (red) expressing mice. n=3, Unpaired Students’ t-test, p=0.003, t(4)=6.418. Scale bar, 25 um. (B) Food intake, measured in grams (g), in control mCherry (black) and hM3Dq (red) expressing Nts-cre mice 1 hour (h), 2h, 4h, 8h and 24h following saline injection. n=5(mCherry, n=5(hM3Dq). Mixed-model ANOVA with post-hoc Bonferroni correction (Time: F(2.33,22.72)=298.8, p<0.0001; Subject: F(1,10)=6.203, p=0.032; Interaction: F(4,39)=1.727, p=0.1637). (C) Food intake, measured in grams (g), in control mCherry (black) and hM3Dq (red) expressing Nts-cre mice 4 hour (h), 8h and 24h following CNO injection. n=7(mCherry), n=10(hM3Dq). Two-way ANOVA with post-hoc Bonferroni correction (Time: F(2,18)=184.5, p<0.0001; Subject: F(1,9)=60.03, p<0.0001; Interaction: F(2,18)=12.38, p=0.0004).(D) Food intake, measured in grams (g), in control mCherry (black) and hM4Di (red) expressing Nts-cre mice 1 hour (h), 2h, 4h, 8h and 24h following saline injection. n=7(mCherry), n=8(hM4Di). Two-way ANOVA with post-hoc Bonferroni correction (Time: F(1,19)=415.8, p<0.0001; Subject: F(1,13)=1.042, p=0.326; Interaction: F(4,52)=2.839, p=0.0333). (E) Food intake, measured in grams (g), in control mCherry (black) and hM4Di (red) expressing Nts-cre mice 4 hours (h), 8h and 24h following CNO injection. n=7(mCherry), n=8(hM3Dq). Two-way ANOVA with post-hoc Bonferroni correction (Time: F(2,18)=184.5, p<0.0001; Subject: F(9,18)=2.945, p=0.0245; Interaction: F(2,18)=12.38, p=0.004).

Figure 2—figure supplement 2
Download asset Open asset
LSNts neurons do not regulate anxiety-like behaviors.

(A) Time, measured in seconds (s), spent in the center and border of an open field in control mCherry (black) and hM3Dq (red) expressing Nts-cre mice. n = 8(mCherry), n = 9(hM3Dq). Two-way ANOVA with post-hoc Bonferroni correction, Subject: F(1,15)=0.071, p=0.794; Arena: F(1,15)=135.8, p<0.0001; Interaction: F(1.15) = 0.9093, p=0.355. (B) Distance, measured in centimeters (cm), traveled in the open field, in control mCherry (black) and hM3Dq (red) expressing Nts-cre mice. n = 13(mCherry), n = 14(hM3Dq), Unpaired Student's t-test, t(25)=0.403, p=0.69. (C) Velocity, measured as the cm per s (cm.s−1), in the open field, in control mCherry (black) and hM3Dq (red) expressing Nts-cre mice. n = 13(mCherry), n = 14(hM3Dq), Unpaired Student's t-test, t(25)=0.915, p=0.369. (D) Time, measured in seconds (s), spent in the center and border of an open field with novelty in control mCherry (black) and hM3Dq (red) expressing Nts-cre mice, n = 5. Two-way ANOVA with post-hoc Bonferroni correction, Subject: F(1,8)=0.358, p=0.566; Arena: F(1,8)=21.54, p=0.0017; Interaction: F(1,8)=0.039, p=0.847. (E) Time, measured in seconds (s), spent in the open and closed arms of an elevated plus maze in control mCherry (black) and hM3Dq (red) expressing Nts-cre mice, n = 5. Two-way ANOVA with post-hoc Bonferroni correction, Arm: F(1,8)=111.2, p<0.0001; Subject: F(1,8)=0.137, p=0.72; Interaction: F(1,8)=1.641, p=0.236. (F) Distance, measured in centimeters (cm), traveled in the elevated plus maze, in control mCherry (black) and hM3Dq (red) expressing Nts-cre mice, n = 5, Unpaired Student's t-test, t(8)=0.5347, p=0.6074. (G) Velocity, measured as the cm per s (cm.s−1), in the elevated plus maze, in control mCherry (black) and hM3Dq (red) expressing Nts-cre mice, n = 5, Unpaired Student's t-test, t(8)=0.533, p=0.608. (H) Latency to feed, measured in seconds (s), in a novelty suppressed feeding task, in control mCherry (black) and hM3Dq (red) expressing Nts-cre mice. n = 5, Unpaired Student's t-test, t(8)=0.027, p=0.979. Data are represented as mean ± SEM.

Figure 3 with 1 supplement
Download asset Open asset
Molecular and functional profiling of LSNts neurons.

(A) Experimental scheme of the viralTrap experiment. Nts-cre mice are injected with an AAV expressing cre-dependent GFPL10 (AAV-Introvert--GFPL10) into the LS and then GFP+ polysomes are immunoprecipitated. (B) Plot depicting the average IP value and average input values (log2) of all genes analyzed. Enriched genes (>2-fold; red) and depleted genes (<2-fold; blue) are shown (q-value <0.05), and selected markers are shown: Mc3r, Glp1r, Nts, Sst, Cartpt, and Mbp (n = 3). All genes depicted have q-values <0.05 as calculated by Cufflinks. (C) Average fold of change (IP/INP Log2) assessed using Taqman qPCR of enriched positive control markers Gfp and Nts, significantly enriched genes Mc3r, Glp1r, Sst, and Cartpt (red) and depleted negative control markers Gfap and Mal (blue) (n = 3 biological replicates). (D) Left panel, representative in situ hybridization image of Nts (green), Vglut2 (red), Vgat (white), and DAPI (blue). Right panel, quantification of Nts cells which express Vgat (white) and Vglut2 (red). n = 3, Unpaired Student's t-test p=0.009; t(4)=4.791. Scale bars, 50 μm. (E) Representative in situ hybridization image of Nts (green) and Glp1r (red). 2× digital zoom is presented in the inset. Scale bars for panels e-h, 50 μm. (F) Representative in situ hybridization image of Nts (green) and Sst (red). Closer magnification is inset. 2× digital zoom is presented in the inset. (G) Representative in situ hybridization image of Nts (green) and Cartpt (red). 2× digital zoom is presented in the inset. (H) Representative in situ hybridization image of Nts (green) and Mc3r (red). 2× digital zoom is presented in the inset. (I) Quantification of the percentage of Nts cells (black) expressing Nts/Glp1r (red), Sst (magenta), Cartpt (blue), and Mc3r (cyan). n = 3(Nts), n = 4(Nts/Glp1r, Nts/Mc3r), n = 6(Nts/Sst), n = 5(Nts/Cartpt) (J) Left panel, experimental scheme. Wild-type mice are injected with exendin-4 (Ex4) directly into the LS or Glp1r-cre mice with viral expression of hM3Dq activatory DREADD receptors are tested for cumulative food intake. (K) Cumulative food intake measured in grams (g) of mice following Vehicle (black) and Ex4 (red) injection in the LS. n = 11(Vehicle), n = 15(Ex4), Two-way ANOVA with post-hoc Bonferroni correction, Time: F(1,24)=100.8, p<0.0001; Treatment: F(1,24)=11.54, p=0.0024; Interaction: F(1,24)=0.002; p=0.963. (L) Cumulative food intake measured in grams (g) of control mCherry (black) and hM3Dq (red) expressing Glp1r-cre mice following CNO injection. Two-way ANOVA with post-hoc Bonferroni correction. n = 10(mCherry), n = 11(hM3Dq). Time: F(1,19)=40.67, p<0.0001; Subject: F(1,19)=20.68, p=0.0002; Interaction: F(1,19)=9.407, p=0.0063. (M) Representative images of Nts (green), Fos (red), and Glp1r (white) in control and restraint stress (RS) mice. Right, quantification of co-localization between Nts, Fos, and Glp1r+ cells in control (black) and RS (red) mice. n = 4, Two-way ANOVA with post-hoc Bonferroni correction, *p<0.05, Row factor: F(1,12)=1.460, p=0.2502, Column Factor: F(1,12)=5.056, p=0.0441, Interaction: F(1.12) = 3.494, p=0.0862. Scale bars, 50 μm. (N) Left, representative in situ hybridization image of Nts (green), Glp1r (blue), and Fos (red) of mice injected with vehicle or the Glp1r agonist Exendin-4 (Ex4). White arrows point to cells co-expressing Nts, Glp1r, and Fos. Right, quantification of Fos+ cells expressing Nts and/or Glp1r in saline (black) or Ex4 (red) injected mice, n = 3(Control), n = 4 (Ex4); Two-way ANOVA with Bonferroni correction, Subject: F(1,10)=71.7, p<0.0001; Treatment: F(1,10)=45.88, p=0.0001; Interaction: F(1,10)=37.58, p<0.0001. Scale bars, 25 μm. Data are represented as mean ± SEM.

Figure 3—figure supplement 1
Download asset Open asset
Regulation of feeding and/or anxiety by Cartpt-, Glp1r- and Sst-expressing neurons in the lateral septum.

(A) Experimental scheme. Wild-type mice are injected with Ex4 or Mc3r agonist y-MSH directly into the LS or Glp1r-cre, Cartpt-Cre, or Sst-cre mice with viral expression of hM3Dq activatory DREADD receptor are tested for cumulative food intake. (B) Food intake, measured in grams (g) of mice 4 hr (h), 12, and 24 hr following Vehicle (black) and Ex4 (red) injection in the LS. n = 11 (Vehicle), n = 15 (Control), Two-way ANOVA with post-hoc Bonferroni correction, Time: F(2,48)=223.6, p<0.0001; Treatment: F(1,24)=9.904, p=0.004; Interaction: F(2,48)=1.67; p=0.199. (C) Food intake, measured in grams (g) of control mCherry (black) and hM3Dq (red) expressing Glp1r-cre mice 4 hr (h), 12, and 24 hr following CNO injection. n = xx, Two-way ANOVA with post-hoc Bonferroni correction. n = 10(mCherry), n = 11(hM3Dq). Time: F(2,38)=296.4, p<0.0001; Subject: F(1,19)=20.32, p=0.0002; Interaction: F(2,38)=6.077, p=0.0051. (D) Food intake, measured in grams (g) of mice 1 hr (h), 2, 4, 12, and 24 hr following vehicle (black) and y-msh (red) injection in the LS. n = 10(vehicle), n = 12(y-msh), Two-way ANOVA with post-hoc Bonferroni correction, Time: F(4,80)=585.6, p<0.0001; Treatment: F(1,20)=0.66, p=0.426; Interaction: F(4,80)=2.67; p=0.038. (E) Food intake, measured in grams (g) of control mCherry (black) and hM3Dq (red) expressing Cartpt-cre mice 1 hr (h), 2, 4, 12, and 24 hr following CNO injection n = 7(mCherry), n = 5(hM3Dq), Two-way ANOVA with post-hoc Bonferroni correction, Time: F(2,17)=1,075, p<0.0001; Subject: F(1,10)=0.1767, p=0.6831; Interaction: F(4,40)=1.364, p=0.2637. (F) Food intake, measured in grams (g) of control mCherry (black) and hM3Dq (red) expressing Sst-cre mice 1 hr (h), 2, 4, 12, and 24 hr following CNO injection, n = xx, Two-way ANOVA with post-hoc Bonferroni correction. n = 11(mCherry), n = 12(hM3Dq). Time: F(4,68)=660.3, p<0.0001; Subject: F(1,17)=3,23, p=0.0903; Interaction: F(4,68)=1.006, p=0.4106. (G) Representative image of viral expression in the lateral septum of Cartpt-cre mice. (H) Representative image of viral expression in the lateral septum of Glp1r-cre mice. (I) Representative image of viral expression in the lateral septum of Sst-cre mice. (J) Time, measured in seconds (s), spent in the open arms and closed arms of an elevated plus maze in control mCherry (black) and hM3Dq (red) expressing Cartpt-cre mice, n = 7(mCherry), n = 5(hM3Dq), Two-way ANOVA with post-hoc Bonferroni correction. Arm: F(1,10)=17.12, p<0.0001; Subject: F(1,10)=1.621, p=0.232; Interaction: F(1,10)=0.02, p=0.891. (K) Time, measured in seconds (s), spent in the open arms and closed arms of an elevated plus maze in control mCherry (black) and hM3Dq (red) expressing Glp1r-cre mice. n = 7(mCherry), n = 8(hM3Dq), Two-way ANOVA with post-hoc Bonferroni correction. Arm: F(1,13)=79.20, p<0.0001; Subject: F(1,13)=0.3198, p=0.5814; Interaction: F(1,13)=0.05114, p=0.825. (L) Time, measured in seconds (s), spent in the open arms and closed arms of an elevated plus maze in control mCherry (black) and hM3Dq (red) expressing Sst-cre mice. n = 8, Two-way ANOVA with post-hoc Bonferroni correction. Arm: F(1,14)=39.48, p<0.0001; Subject: F(1,14)=0.0007, p=0.979; Interaction: F(1,14)=0.1811, p=0.677. Scale bars (G-I) are 50 μm. Data are represented as mean ± SEM.

Figure 4 with 1 supplement
Download asset Open asset
LSNts- > LH projection regulates food intake in mice.

(A) Representative images of the LS injection site (left) and terminals in the lateral hypothalamus (left) of Nts-cre mice injected with a control mCherry reporter virus. Atlas images above the representative images depict the area of magnification. Scale bar, 50 μm. (B) Representative image of cells labeled in the lateral hypothalamus (right) of Nts-cre mice injected with the anterograde tracer H129ΔTK-tdTomato in the LS. Middle, atlas image depicting the area of magnification to the right. Scale bar, 25 μm. (C) Left, experimental scheme depicting mice injected with AAV expressing Chr2 or control mCherry into the LS and an optic fiber positioned over the lateral hypothalamus. Middle, food intake was measured in an OFF-ON-OFF paradigm. Right, food intake measured in grams (g) of control mCherry (black) or Chr2 (red) expressing mice before (OFF), during (ON) and after (OFF) laser stimulation. n = 11, Two-way ANOVA with post-hoc Bonferroni correction, Epoch: F(2,40)=38.47, p<0.0001; Subject: F(1,20)=0.069, p=0.796; Interaction: F(2,40)=10.94, p=0.0002. Data are represented as mean ± SEM.

Figure 4—figure supplement 1
Download asset Open asset
LSNts → LH does not regulate anxiety or locomotion.

(A) Left, experimental scheme of Glp1r-cre mice injected with AAV expressing cre-dependent mCherry in the lateral septum. Right, representative image of Glp1r fibers in the lateral hypothalamus. Scale bars are 100 μm. (B) Left, experimental scheme of Nts-cre mice injected with AAV expressing Chr2 in the lateral septum and optic fibers in the lateral hypothalamus. Right, Representative image of fiber optic path in the lateral hypothalamus. Scale bars are 100 μm. (C) Left, representative images of c-fos (green) and mCherry (red) in control mCherry (left) and Chr2 (right) expressing Nts-cre mice injected with CNO. Right, quantification of c-fos+ cells in control (black) and Chr2 (red) mice. n = 3, Unpaired Student's t-test, t(4)=4.999, p=0.008. Scale bar, 50 μm. (D) Left, representative images of c-fos (red) and mCherry projections (green) in the lateral hypothalamus of Nts-cre mice expressing control mCherry (left) or Chr2 (right) virus injected with CNO. n = 3, Unpaired Student's t-test, t(4)=5.089, p=0.007. Scale bar, 50 μm.(E) Distance, measured in centimeters (cm), traveled in the elevated plus maze, in control mCherry (black) and Chr2 (red) expressing Nts-cre mice. n = 5, Unpaired Student's t-test, t(8)=0.9092, p=0.39. (F) Velocity, measured as the cm per s (cm.s−1), in the elevated plus maze, in control mCherry (black) and Chr2 (red) expressing Nts-cre mice. n = 5, Unpaired Student's t-test, t(8)=1.291, p=0.233. (G) Time, measured in seconds (s), spent in the center and border of an open field in control mCherry (black) and Chr2 (red) expressing Nts-cre mice. n = 5. Two-way ANOVA with post-hoc Bonferroni correction, Subject: F(1,8)=0.051, p=0.827; Border/Center: F(1,8)=18.48, p=0.0026; Interaction: F(1,8)=0.791, p=0.4. Data are represented as mean ± SEM.

Figure 5 with 1 supplement
Download asset Open asset
LSNts neurons are specifically activated by stressful situations involving active coping.

(A) Left panel, experimental scheme: mice were manually immobilized for the period of 1 min. Ca+2 activity was recorded before, during, and after the period of manual immobilization. Middle, Average GCaMP6s z-score (black lines) and SEM (gray dotted lines) across all recordings and time-locked to immobilization start. Right middle panel, quantification of the area under the curve of Ca+2 transients before, during, and after immobilization. One-way ANOVA with repeated measures and post-hoc Bonferroni correction, F(1.833, 9.165)=9.888, p=0.0057. Right panel, heat maps represent the average % ΔF/F from each GCaMP6s recordings time-locked to immobilization start. n = 6. (B) Left panel, experimental scheme: mice were tail suspended for the period of 1 min. Ca+2 activity was recorded before, during, and after the period of tail suspension. Middle, average GCaMP6s z-score (black lines) and SEM (gray dotted lines) across all recordings and time-locked to tail suspension start. Right middle panel, quantification of the area under the curve of Ca+2 transients before, during, and after tail suspension. Paired Student’s t-test, t = 2.659, df = 5. Right panel, heat maps represent the average % ΔF/F from each GCaMP6s recordings time-locked to tail suspension start. n = 6. (C) Left panel, experimental scheme: mice were exposed to an open field arena containing a robotic, remote-controlled spider predator and a designated safe ‘nest’ (black triangular hut). Ca+2 activity was recorded during the whole session. Middle, average GCaMP6s z-score (black lines) and SEM (gray dotted lines) across all recordings and time-locked to the robotic spider attack. Right middle panel, quantification of the area under the curve of Ca+2 transients before and after the robotic spider attack. Paired Student’s t-test, t = 3.507, df = 4. Right panel, heat maps represent the average % ΔF/F from each GCaMP6s recordings time-locked to the robotic spider attack. n = 5. (D) Left panel, experimental scheme: the designated safe ‘nest’ (black triangular hut) was removed and mice that had been attacked by a robotic spider predator was exposed to the open field containing the spider. Ca+2 activity was recorded during the whole session. Middle, average GCaMP6s z-score (black lines) and SEM (gray dotted lines) across all recordings and time-locked to the removal of the nest. Right middle panel, quantification of the area under the curve of Ca+2 transients before and after the removal of the nest. Paired Student’s t-test, t = 3.305, df = 5. Right panel, heat maps represent the average % ΔF/F from each GCaMP6s recordings time-locked to the removal of the nest. n = 6. (E) Experimental scheme for contextual fear conditioning: mice were exposed to an operant chamber where they received a mild foot shock (training) and after 24 hr mice were re-exposed to the operant chamber (testing). Ca+2 activity was recorded during training and testing. (F) First panel, average GCaMP6s z-score (black lines) and SEM (gray dotted lines) across all recordings and time-locked to the foot shock received on the training day. Second panel, quantification of the area under the curve of Ca+2 transients before and after the foot shock. Paired Student’s t-test, t = 3.230, df = 4. Third panel, heat maps represent the average % ΔF/F from each GCaMP6s recordings time-locked to the freezing behavior. n = 6. (G) First panel, average GCaMP6s z-score (black lines) and SEM (gray dotted lines) across all recordings and time-locked to freezing behavior on the testing day. Second panel, quantification of the area under the curve of Ca+2 transients before and after the freezing behavior. Paired Student’s t-test, t = 2.833, df = 8. Third panel, heat maps represent the average % ΔF/F from each GCaMP6s recordings time-locked to the freezing behavior. n = 6. Data are represented as mean ± SEM.

Figure 5—figure supplement 1
Download asset Open asset
LSNts neurons are not selectively tuned to feeding or movement.

(A) Experimental scheme. Mice were then attached to a fiber photometry setup with a 465 nm LED (blue) and a 405 nm LED (purple) and a photodetector. (B) Representative image of viral expression in the LS of Nts-cre mice injected with GCaMP6s and implanted with an optic fiber in the LS. Scale bar, 50 μm. (C) Average GCaMP6s z-score (black lines) and SEM (gray dotted lines) across all recording sites time-locked to the start of the feeding bout. Right, heat maps represent the average % DF/F from each GCaMP6s recordings time-locked to feeding bout start. (D) Area under the curve (a.u.) of the fluorescent signal before (black) and after (red) the feeding bout start. n = 6, Paired Student's t-test, t(5)=1.304, p=0.249. (E) Average GCaMP6s z-score (black lines) and SEM (gray dotted lines) across all recording sites time-locked to the start of locomotion. (F) Area under the curve (a.u.) of the fluorescent signal before (black) and after (red) the start of locomotion. n = 5, Paired Student's t-test, t(4)=0.402, p=0.7083. (G) Experimental scheme of contextual fear conditioning. (H) Percentage of freezing in Context A (CS+, red) and Context B (CS−, black). Unpaired Student’s t-test, *p<0.05, t = 3.233, df = 4, n = 3. Data are represented as mean ± SEM.

Videos

Video 1
Download asset
Representative video of the simulated ‘predator’ test.

The mouse is placed in the open field with a designated safe zone. A robotic spider ‘predator’ is introduced and ‘attacks’ as the mouse exits the safe zone. Calcium recordings were taken throughout the entire task.

Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)Mouse: Nts-creJackson LaboratoriesStock#017525N/A
Genetic reagent (Mus musculus)Mouse: Cartpt-creJackson LaboratoriesStock#028533N/A
Genetic reagent (Mus musculus)Mouse: Sst-creJackson LaboratoriesStock#013044N/A
Genetic reagent (Mus musculus)Mouse: Glp1r-creJackson LaboratoriesStock#029283N/A
AntibodyAnti-phospho ribosomal protein 6 pSer244/pSer247 (Rabbit polyclonal)InvitrogenCat#44–923G, RRID:AB_2533798(1:1000)
AntibodyAnti-GFP (Chicken polyclonal)AbcamCat#ab13970, RRID:AB_300798(1:1000)
Antibody19C8 and 19F7, GFP monoclonal antibodiesMemorial Sloan-Kettering Monoclonal Antibody FacilityCustom order(50 ug)
AntibodyAnti-mCherry (Chicken polyclonal)AbcamCat# ab205402(1:1000)
AntibodyMouse anti-cfos (Rabbit monoclonal)Cell SignalingCat#2250S, RRID:AB_2247211(1:500)
AntibodyAnti-rabbit IgG Alexa 488 (Goat polyclonal)InvitrogenCat#A11008, RRID:AB_143165(1:1000)
AntibodyAnti-rabbit IgG Alexa 594 (Goat polyclonal)InvitrogenCat#A11072, RRID:AB_142057(1:1000)
AntibodyAnti-chicken IgG Alexa 488 (Goat polyclonal)InvitrogenCat#A11039, RRID:AB_142924(1:1000)
AntibodyAnti-chicken IgG Alexa 594 (Goat polyclonal)InvitrogenCat#A11042, RRID:AB_142083(1:1000)
Recombinant DNA reagentAAV5-EF1a-DIO-YFPUNC Vector CoreN/AN/A
 recombinant DNA reagentAAV5-EF1a-DIO-hChR2(H134R)-YFPUNC Vector CoreN/AN/A
Recombinant DNA reagentAAV5-EF1a-DIO-hM3D(Gq)-mCherryUNC Vector CoreN/AN/A
Recombinant DNA reagentAAV5-EF1a-DIO-mCherryUNC Vector CoreN/AN/A
Recombinant DNA reagentAAV5-EF1a- DIO-hM4D(Gi)-mCherryUNC Vector CoreN/AN/A
Recombinant DNA reagentAAV5-DIO-GCaMP6sAddgeneCat#510882N/A
Recombinant DNA reagentHSV-lsl-tdTomatoLo and Anderson, 2011N/AN/A
Recombinant DNA reagentAAV5-Introvert-GFPL10aNectow et al., 2017N/AN/A
Sequence-based reagentPrimeTime Standard qPCR Assay for NTSidtDNAMm.PT.58.10351472N/A
Sequence-based reagentPrimeTime Standard qPCR Assay for ActBidtDNAMm.PT.47.5885043.gN/A
Sequence-based reagentProbe for NTS (C1)ACDBioCat# 420448N/A
Sequence-based reagentProbe for cFos (C2)ACDBioCat#506921-C2N/A
Sequence-based reagentProbe for Glp1r (C3)ACDBioCat#418851-C3N/A
Sequence-based reagentProbe for Sst (C3)ACDBioCat#404631-C3N/A
Sequence-based reagentProbe for Mc3r (C3)ACDBioCat#412541-C3N/A
Sequence-based reagentProbe for Cartpt (C3)ACDBioCat#432001-C3N/A
Sequence-based reagentProbe for vGAT (C2)ACDBioCat#319191-C2N/A
Sequence-based reagentProbe for vGLUT2 (C3)ACDBioCat#319171-C3N/A
Peptide, recombinant proteinNBL10 recombinant peptideChromotekCat# GT-250100 ng/mL
Commercial assay or kitRNAeasy Mini kitQiagenCat#74104N/A
Commercial assay or kitRNAscope Fluorescent Multiplex Reagent Kit V2ACDbioCat#323100N/A
Chemical compound, drugClozapine-N-oxide(CNO)Tocris BiosciencesCat#4936; CAS: 34233-69-71 mg/kg
Software, algorithmTophatBasespacehttps://basespace.illumina.com/apps/N/A
Software, algorithmCufflinksBasespacehttps://basespace.illumina.com/apps/N/A
Software, algorithmSYNAPSETucker-Davis Technologieshttps://tdt.comN/A
Software, algorithmMATLABMathworkshttps://mathworks.comN/A
Software, algorithmImageJNIHhttps://imagej.nih.gov/ij/N/A
Software, algorithmGraphPad Prism 5.0GraphPadhttps://www.graphpad.com/scientific-software/prism/N/A
Software, algorithmEthovision 9.0Noldushttps://noldus.comN/A

Additional files

Supplementary file 1

c-Fos expression in the mouse brain after restraint stress.

Expression is noted as − (no expression), + (low expression), ++ (moderate expression) and +++ (high expression).

https://cdn.elifesciences.org/articles/58894/elife-58894-supp1-v1.docx
Supplementary file 2

Phospho-TRAP differential gene expression data.

Data is sorted by fold-change (log2) in restraint stress (RS) samples compared to Naïve (N) samples along with the corresponding q-values.

https://cdn.elifesciences.org/articles/58894/elife-58894-supp2-v1.xlsx
Supplementary file 3

Viral-TRAP differential gene expression data.

Significantly enriched (TRUE) genes are shown and are sorted by fold-change (log2).

https://cdn.elifesciences.org/articles/58894/elife-58894-supp3-v1.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/58894/elife-58894-transrepform-v1.pdf

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Estefania P Azevedo
  2. Bowen Tan
  3. Lisa E Pomeranz
  4. Violet Ivan
  5. Robert Fetcho
  6. Marc Schneeberger
  7. Katherine R Doerig
  8. Conor Liston
  9. Jeffrey M Friedman
  10. Sarah A Stern
(2020)
A limbic circuit selectively links active escape to food suppression
eLife 9:e58894.
https://doi.org/10.7554/eLife.58894