Abstract
Neural networks that control reproduction must integrate social and hormonal signals, tune motivation, and coordinate social interactions. However, the neural circuit mechanisms for these processes remain unresolved. The medial preoptic area (mPOA), an essential node for social behaviors, comprises molecularly diverse neurons with widespread projections. Here we identify a steroid-responsive subset of neurotensin (Nts)-expressing mPOA neurons that interface with the ventral tegmental area (VTA) to form a socially engaged reward circuit. Using in vivo two-photon imaging in female mice, we show that mPOANts neurons preferentially encode attractive male cues compared to nonsocial appetitive stimuli. Ovarian hormone signals regulate both the physiological and cue-encoding properties of these cells. Furthermore, optogenetic stimulation of mPOANts–VTA circuitry promotes rewarding phenotypes, social approach and striatal dopamine release. Collectively, these data demonstrate that steroid-sensitive mPOA neurons encode ethologically relevant stimuli and co-opt midbrain reward circuits to promote prosocial behaviors critical for species survival.
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Change history
30 June 2017
In the version of this article initially published, there were errors in data analysis and presentation. The corrected analysis and presentation do not change the results or interpretation of the data. Asterisk definitions have also been added for clarity as noted below. Changes with respect to the number of subjects reflect errors in reporting only and did not affect the data analysis. The percentages in Figure 1b originally reported as 43, 3, 18 and 36% have been changed to 43, 1, 19 and 37%, respectively. In the first paragraph of the Results, “97% of Nts-labeled cells colocalizing with VTA beads” has been changed to “96% of Nts-labeled cells colocalizing with VTA beads” and “this subpopulation comprised 33% of all mPOA VTA-projecting neurons” has been changed to “this subpopulation comprised 35% of all mPOA VTA-projecting neurons.” In the legend for Figure 2a, the n value originally reported as 9 has been changed to 9 and 10, and asterisks have been added to read ***P = 0.0006. In Figure 3a, points were misplotted as a result of an error in data analysis. The graph has been replaced. The values originally reported in the legend as t5 = 2.82, P = 0.0368 have been changed to t5 = 5.85, **P = 0.0021. In the Figure 3d legend, the value originally reported as n = 52 cells has been changed to 51. In Figure 3e, the percentage in the male E2 pie chart for excited neurons has been changed from 24 to 23%. Figure 5b originally contained duplicate example traces of calcium transients that were supposed to be taken from three individual neurons; new traces have been supplied. In Figure 5c,d, the asterisks have been changed from *** to **** and defined in the legend as ****P < 0.0001, Bonferroni post hoc test, E2 versus pre and E2 versus post. In the Figure 5d legend, the value originally reported as F2,252 = 13.13 has been changed to 17.32. In the Figure 5f legend, asterisks have been defined as ***P < 0.001, Bonferroni post hoc test. In Figure 5g, the horizontal axis was truncated at 60, resulting in missing data points; the graph has been replaced. In the Figure 5h legend, asterisks have been defined as *P < 0.05, **P < 0.01, ***P < 0.001, Bonferroni post hoc test. In the Figure 6e legend, the degrees of freedom originally reported as F3,30 have been changed to F3,27. In the Figure 6g legend, the value originally reported as eYFP = 6 mice has been changed to eYFP = 7 mice. In the Figure 6h legend, the values originally reported as F3,30 = 9.44, P = 0.0002 have been changed to F3,24 = 15.2, P < 0.0001. In Figure 6i,j, points were misplotted as a result of an error in data analysis, and error bars plotted as s.d. were misidentified in the legend as s.e.m. The data have been replotted, with error bars representing s.e.m. In the Figure 6i legend, the values originally reported as F1,8 = 23.2 have been changed to F1,9 = 63.1. Asterisks have been defined at the end of the Figure 6 legend as *P < 0.05, ***P < 0.001, ****P < 0.0001, Bonferroni post hoc test. The statistically significant differences in Figure 7b originally indicated by ** have been changed to ***; these have been defined in the legend as ***P < 0.001, Bonferroni post hoc test. In Figure 7e, a data point in the E2 group was missing; the graph has been replaced. An asterisk has also been added in the Figure 7e legend to read *P = 0.016. In Figure 8c, data were misplotted as a result of errors in analysis. The graph has been replaced, and the statistically significant differences originally indicated by * have been changed to **. The values originally reported in the legend as F4,24 = 4.20, P = 0.0112 have been changed to F4,21 = 6.82, P = 0.0011. In Figure 8d, data were misplotted as a result of an error in analysis. The graph has been replaced, and the values originally reported in the legend as F4,24 = 8.33, P = 0.0003 have been changed to F4,21 = 6.35, P = 0.0016. In Figure 8e, data were misplotted as a result of an error in analysis. The graph has been replaced, and the values originally reported in the legend as F4,24 = 0.60, P = 0.6622 have been changed to F4,21 = 1.33, P = 0.29. In Figure 8g, the vertical axis was truncated at 250, resulting in missing data points; the graph has been replaced, and the statistically significant differences originally indicated by * have been changed to **. The values originally reported in the legend as F1,12 = 7.92 as a result of an error in manuscript preparation have been changed to F1,12 = 7.15. In Figure 8h, the vertical axis was truncated at –0.4 and 0.8, resulting in missing data points, and data were misplotted as a result of an error in analysis. The graph has been replaced, and the statistically significant differences originally indicated by * have been changed to ***. The values originally reported in the legend as F1,12 = 7.15, P = 0.0200 have been changed to F1,12 = 9.8, P = 0.009. In Figure 8j, the vertical axis was truncated at 200, resulting in missing data points. The graph has been replaced, and the test statistic originally described in the legend as t3,33 has been changed to F3,33. In Figure 8k, the vertical axis was truncated at –0.4 and 0.4, resulting in missing data points, and data were misplotted as a result of an error in analysis. The graph has been replaced, and the statistically significant differences originally indicated by *** have been changed to **. The value originally reported in the legend as n = 7 has been changed to control n = 6 and NpHR n = 7. In Supplementary Figure 5b–d,f,g, points were misplotted as a result of errors in data analysis and figure preparation; the graphs have been replaced. The values originally reported in the legend to Supplementary Figure 5b as t1,11 = 1.71 p = 0.1042, n = 8 mice have been changed to t1,9 = 1.09 p = 0.3061, n = 10 mice. The values originally reported in the legend to Supplementary Figure 5c–e as n = 8 have been changed to n = 7-10. The values originally reported in the legend to Supplementary Figure 5f as F4,37 = 3.61, p = 0.0137, n = 8 mice have been changed to F4,37 = 3.34, p = 0.0196, n = 7-10 mice. The values originally reported in the legend to Supplementary Figure 5g as F4,37 = 1.74, p = 0.1621, n = 8 mice have been changed to F4,37 = 2.01, p = 0.1131, n = 7-10 mice. In Supplementary Figure 6c–e, points were misplotted as a result of an error in data analysis; the graphs have been replaced. The values originally reported in the legend to Supplementary Figure 6c as F2,15 = 17.03, p = 0.0002 have been changed to F2,17 = 35.61, p = 0.0003, and the statistically significant differences originally indicated by *** for Veh have been changed to ** for E2 and *** for E2-P4, with asterisks defined in the legend as Veh. vs. E2, **P = 0.004; Veh. vs. P4, ***P = 0.0003. The values originally reported in the legend to Supplementary Figure 6d as F2,15 = 11.32, p = 0.0010 have been changed to F2,17 = 27.86, p = 0.0008, and the statistically significant differences originally indicated by * for E2 have been changed to **, with asterisks defined in the legend as Veh. vs. E2, **P = 0.009; Veh. vs. P4, ***P = 0.00019. The values originally reported in the legend to Supplementary Figure 6e as F2,15 = 00.48, p = 0.6268 have been changed to F2,17 = 1.76, p = 0.238. The values originally reported in the legend to Supplementary Figure 10b–f as n = 6 have been changed to n = 5-6. The values originally reported in the legend to Supplementary Figure 10c as F4,26 = 1.04 p =.09322 have been changed to F4,26 = 0.21 p = 0.9322. The degrees of freedom originally reported in the legend to Supplementary Figure 10f as F2,15 have been changed to F2,13. The test description and values originally reported in the legend to Supplementary Figure 10i as One-Way ANOVA, interaction F2,13 = 0.72 p = 0.5018 have been changed to One-Way ANOVA, F2,17 = 0.15 p = 0.8640. The test name and values originally reported in the legend to Supplementary Figure 10j as One-Way ANOVA, interaction F2,22 = 9.09 p = 0.9099, n = 7 have been changed to Two-Way ANOVA, interaction F2,22 = 0.47 p = 0.9543, n = 6-7. The values originally reported in the legends to Supplementary Figure 10k,l as n = 7 have been changed to n = 6-7. The test name and values originally reported in the legend to Supplementary Figure 10m as One-Way ANOVA, interaction F2,22 = 1.29 p = 0.2953, n = 7 have been changed to Two-Way ANOVA, interaction F2,22 = 0.69 p = 0.5142, n = 6-7. The values originally reported in the legend to Supplementary Figure 10n as One-Way ANOVA, interaction F2,22 = 0.686 p = 0.5142, n = 7 have been changed to Two-Way ANOVA, interaction F2,22 = 0.405 p = 0.6719, n = 6-7. The test name and values originally reported in the legend to Supplementary Figure 10o as Paired t-test, t1,10 = 0.34 p = 0.7395, n = 8 have been changed to un-paired t-test, t1,10 = 0.70 p = 0.4983, n = 6. The errors have been corrected in the HTML and PDF versions of the article. Images of the original figures alongside the revised figures are shown in the correction notice appended to the PDF version of the article.
30 June 2017
In the version of this article initially published, images were pixelated as a result of the production processes used to create the online version. The images have been replaced in the HTML and PDF versions of the article.
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Acknowledgements
We thank the Stuber laboratory for discussion and support. We thank S. Smith for advice on two-photon imaging, the UNC vector core for viral packaging, and the UNC Neuroscience Center Microscopy Core, especially V. Ghukasyan, for his training and assistance (P30 NS045892). We thank K. Deisseroth for viral constructs (Stanford University). We thank L. Tarantino and S. Schoenrock for their input and assistance with hormone manipulation procedures. We thank K. Schilling-Scrivo, L. Eckman, J. Rodriguez and G. Tipton for technical assistance and R. Ung for assistance with coding. J.A.M. was supported by the National Institute of Mental Health (T32-MH093315), J.M.O. was supported by the National Institute on Drug Abuse (F32-DA041184), Z.A.M. was supported by ABMRF and NIAAA (K01 AA020911), J.E.R. was supported by NIAAA (F30AA021312) and M.A.R. was supported by NIDDK (F32-DK112564). This study was supported by funds from the Foundation of Hope, the Brain and Behavior Research Foundation, the Simons Foundation, the National Institute on Drug Abuse (R01 DA032750 and R01 DA038168) (G.D.S.), the Department of Psychiatry at UNC-CH, and the National Institute on Alcohol Abuse and Alcoholism (AA022449) (E.A.B.).
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J.A.M. and G.D.S. designed the experiments and wrote the manuscript with input from all authors. J.M.O. and J.E.R. performed physiology recordings. E.A.B. performed fast-scan cyclic voltammetry. M.A.R. wrote code for in vivo imaging data analysis. O.K. and Z.A.M. performed in situ hybridization. N.W.M. assisted with tissue processing. J.A.M. performed all surgeries, behavioral assays, two-photon and confocal imaging experiments. D.R.R. and G.D.S. supervised the project.
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The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Validation of VTA placement and nuclear ESR1 protein expression.
a. Schematic illustrates Retrobead tracer injection into the VTA and DIO-eYFP injection into the mPOA. VTA: ventral tegmental area, mPOA: medial preoptic area.
b. Confocal image of Retrobead injections into the VTA from a representative animal. PAG: periaqueductal gray, D: dorsal, V: ventral, M: medial, L: lateral, scale bar: 100 μm.
c. Confocal image of Nts Ai9 reporter expression (blue) and ESR1-ir (pink). ESR1: estrogen receptor 1.
d. Confocal image of Nts labeled neurons and nuclear ESR1-ir in the mPOA scale bar: 40 μm.
Supplementary Figure 2 Expression of Vgat, Vglut2 and Nts in mPOA neurons.
a. Confocal image of Nts (green) and Vgat (blue) mRNA in the mPOA (left). Scale bar: 200 μm. Color dot plot from a representative brain slice illustrates the X-Y coordinates of each Vgat expressing cell that is also positive (green) or negative (blue) for Nts (right hemisphere). Scale bar: 500 μm, Nts: neurotensin, Vgat: vesicular GABA transporter, mPOA: medial preoptic area, vBNST: ventral bed nucleus of the stria terminalis, D: dorsal, V: ventral, M: medial, L: lateral, AC: anterior commissure, OX: optic chiasm.
b. Confocal image of Nts (green) and Vglut2 (pink) mRNA in the mPOA (left). Scale bar, 200 μm. Color dot plot from a representative subject illustrates the X-Y coordinates of each Vglut2 expressing cell that is positive (green) or negative (pink) for Nts. Scale bar, 500 μm, Vglut2: vesicular glutamate transporter, for remaining abbreviations refer above (A).
c. Confocal image of Nts (green), Vgat mRNA (blue), and Vglut2 mRNA (pink) mRNA in the mPOA. Scale bar: 20 μm.
d. Cumulative distribution plot showing that the mean intensity of Nts expression within each cell that is positive for Nts is higher in estradiol treated mice compared to vehicle treated controls (Kolmogorov-Smirnov D = 0.53, p < 0.0001, 1,900 cells, n = 3 mice per group).
e. Cumulative distribution plot showing that the mean intensity of Vgat expression within each cell that is positive for Nts is higher in estradiol treated mice compared to vehicle treated controls (Kolmogorov-Smirnov D = 0.35, p < 0.0001, 1,900 cells, n = 3 mice per group).
f. Cumulative distribution plot showing that the mean intensity of Vgat expression within each cell that is negative for Nts is higher in estradiol treated mice compared to vehicle treated controls (Kolmogorov-Smirnov D = 0.11, p < 0.0001, 1,900 cells, n = 3 mice per group).
g. Cumulative distribution plot showing that the mean intensity of Vglut2 expression within each cell that is negative for Nts is higher in estradiol treated mice compared to vehicle treated controls (Kolmogorov-Smirnov D = 0.23, p < 0.0001, n = 3 mice per group).
h. Pie chart illustrates the percentage of Nts neurons co-expressing Vgat and/or Vglut2.
Supplementary Figure 3 Validation of Cre and Nts in mPOA-Nts-IRES mice.
a. Representative confocal image of Nts mRNA (green) in the mPOA. mPOA: medial preoptic area, vBNST: ventral bed nucleus of the stria terminalis, OX: optic chiasm, scale bar: 200 μm.
b. Representative confocal image of Cre mRNA (magenta) in the mPOA.
c. Merged confocal image of Nts mRNA (green) and Cre mRNA (magenta).
d. Representative confocal image of Nts mRNA (green) and Cre mRNA (magenta) in the mPOA. Scale bar: 40 μm.
e. Quantification of Nts mRNA (green) and Cre mRNA (magenta) overlap in the mPOA. Pie chart represents overlap.
Supplementary Figure 4 Validation and implementation of mPOANts in vivo calcium imaging
a. Schematic represents whole cell patch clamp recordings in an mPOANts::GCaMP6 neuron while simultaneously imaging Ca2+ activity. Representative cell shows that increasing current injection increased the intensity of Ca2+ fluorescence.
b. Example calcium traces, F/F0 during patch clamp recordings in mPOANts::GCaMP6 cells from ovariectomized mice treated with vehicle (vehicle) or estradiol (E2) primed 48 hours and 4 hours prior to collecting slices.
c. Estradiol enhanced the Z-score of normalized peak response from baseline in mPOANts::GCaMP6 cells, compared to controls, and increasing current injection increased z-score. (Two-Way ANOVA, interaction F8,104 = 4.47, p < 0.0001, Veh n= 6 cells, E2 n= 9 cells; 2 mice per group).
d. Schematic depicts surgical and in vivo imaging approach. Cre-inducible GCaMP6s in mPOANts neurons and Gradient Refractive Index (GRIN) lens for two-photon optical access in awake head-fixed mice.
e. Image represents a standard deviation z-projection acquired from a motion corrected two-photon baseline recording from mPOANts::GCaMP6. Images were used for hand drawing regions of interests (ROIs) over cells.
f. Illustration is an overlay representing hand drawn regions of interest (ROIs) onto a standard deviation projection (c) for subsequent somatic Ca2+ signal extraction in individual neurons.
Supplementary Figure 5 Behavioral preference for attractive odors.
a. Schematic illustrates test for behavioral odor preference for an odor following a habituation to an empty arena.
b. Female mice had no side preference during habituation periods that occurred prior to odor testing (paired t-test, t1,9 = 1.09 p = 0.3061, n = 10 mice).
c. Nose-point time was highest during odor trials consisting of intact male urine, compared to all other odorants. (One-way Anova, F4,37 = 10.60 p < 0.0001, n = 7-10 mice).
d. There was no difference in the amount of nose-point time in the saline control zone during each odor test. (One-way Anova, F4,37 = 1.05 p = 0.3950, n = 7-10 mice).
e. All odors induced a behavioral preference for the odor zone over the saline control zone and the preference index for male urine was highest than that of gonadectomzied male or female urine. (One-way Anova, F4,37 = 5.38, p = 0.0016, n = 7-10 mice).
f. Females had the highest odor zone approach bouts to male urine. (One-way Anova, F4,37 = 3.34, p = 0.0196, n = 7-10 mice).
g. Females did not display differences in saline approach bouts. (One-way Anova, F4,37 = 2.01, p = 0.1131, n = 7-10 mice).
Supplementary Figure 6 Behavioral and neuronal responses across hormonal states.
a. Schematic illustrates steroid-priming of estradiol (E2) and subsequently progesterone (P4) 4-6 hours prior to behavioral odor testing and compared with an oil injection (vehicle).
b. Heat maps of total time spent in an odor zone from a representative female before and after estradiol priming.
c. Ovariectomzied females had a higher preference for male odor following estradiol priming that was sustained following progesterone administration, with no differences between steroid treatments (One-way Anova, F2,17 = 35.61, p = 0.0003, n = 6 mice). Veh. vs. E2, **P = 0.004; Veh. vs. P4, ***P = 0.0003.
d. Ovariectomzied females spent more time in the odor zone following estradiol priming and this was sustained following progesterone administration, with no differences between steroid treatments (Oneway Anova, F2,17 = 27.86, p = 0.0008, n = 6 mice). Veh. vs. E2, **P = 0.009; Veh. vs. P4, ***P = 0.00019.
e. No treatment differences were detected in the amount of time spent investigating the control block. (One-way Anova, F2,17 = 1.76, p = 0.238, n = 6 mice).
f. Ca2+ signal normalized to baseline and averaged across male trials. Heat map illustrates response from the same 230 mPOANts::GCaMP6 cells across three separate imaging days. Cells are sorted by male odor response on the day of E2 administration (middle heat plot) and indexed in the same order for comparison on the day of Veh administration (left heat plot) and after P4 administration (right heat plot).
g. Top: Pie charts illustrate the percentage of cells excited (green) or inhibited (blue) in response to male odor by treatment. Bottom: Averaged Ca2+ traces from mPOANts::GCaMP6 cells significantly excited by male odor after estradiol, compared to their response after vehicle or progesterone (Wilcoxon t-test, all p values < 0.05, n = 230 cells combined across 4 mice).
h. Ca2+ signal normalized to baseline and averaged across female trials. Heat map illustrates response from the same 230 mPOANts::GCaMP6 cells across two separate imaging days. Cells are sorted by female odor response on the day of estradiol administration (right heat plot) and indexed in the same order for comparison on the day of Veh administration (left heat plot).
i. mPOANts::GCaMP6 cells imaged across 2 days of the estrous cycle. Arrows refer to examples of the same cells.
j. Example Ca2+ traces, F/F0, of GCaMP6s Ca2+ dynamics from mPOANts::GCaMP6 cells across two days of the estrous cycle. Arrows point out examples of the same cells across days.
k. mPOANts::GCaMP6 cells had Ca2+ events longer in duration on proestrus compared to estrus (Paired t-test, t154= 2.26, p = 0.0255, n = 155 cells combined across 3 mice).
l. mPOANts::GCaMP6 cells had calcium events higher in peak amplitude on proestrus compared to estrus (Paired t-test, t154= 2.56, p = 0.0113, n = 155 cells combined across 3 mice.
Supplementary Figure 7 Photostimulation of mPOANts in male and female mice.
a. Estrous cycle cytology representing the four days of the mouse estrous cycle. P: proestrus, E: estrus, DI: metestrus, DII: diestrus, scale bar, 62 μm.
b. Individual number of nose pokes into the active port from mPOANts::ChR2 females during proestrus and estrus. Regardless of cycle, differences were observed in the number of pokes as a function of frequency, with differences detected between all frequencies except for between 5 and 10 Hz (Two-Way ANOVA, frequency, F3,18 = 199, p < 0.0001, ChR2 n = 4 mice).
c. mPOANts::ChR2 females poked more during 20 and 40 Hz (but not 5 or 10 Hz) of photostimulation on proestrous compared to estrous. (Two-Way ANOVA, interaction, F3,18 = 14.10, p < 0.0001, ChR2 n = 4 mice).
d. mPOANts::ChR2 females displayed a significantly higher mean velocity in the stimulation side of the chamber in all phases of the estrous cycle compared to mPOANts::eYFP controls (Two-Way ANOVA, interaction F3,30 = 5.75, p = 0.0031, eYFP n = 7 mice, ChR2 n = 5 mice).
e. mPOANts::ChR2 males displayed a significantly higher mean velocity on the stimulation side of the chamber compared to mPOANts::eYFP controls with no effect of day (Two-Way ANOVA, group effect, F1,8 = 31.10, p = 0.0008, eYFP n = 6 mice, Chr2 n = 4 mice).
f. Location of optical fiber placements in the mPOA from mPOANts male and female optogenetic cohorts.
Supplementary Figure 8 Photostimulation of mPOANts neurons induces Fos in the mPOA.
a. Confocal image from an mPOANts::ChR2 mouse (top) and mPOANts::eYFP control (bottom). vBNST= ventral bed nucleus of the stria terminalis. D: dorsal, V: ventral, M: medial, L: lateral, AC: anterior commissure, OX: optic chiasm, scale bar: 200 um.
b. Confocal image of mPOANts::ChR2 (green) and optically-evoked Fos immunoreactivity (red), scale bar: 50 um (left column).
c. 20 Hz photostimulation increased the number of cells expressing Fos immunoreactivity in mPOANts::ChR2 mice compared to mPOANts::eYFP controls (Unpaired t-test t4 = 15.87, p < 0.0001, n = 3 mice per group).
Supplementary Figure 9 Optogenetic modulation of mPOANts neurons during hormonal and social contexts.
a. Top: Schematic illustrates testing paradigm. Females were tested for real-time place preference after a vehicle control injection once a week preceding and following steroid priming. Steroid-priming consisted of an injection of estradiol and progesterone 48 hours apart, with testing 4 hours after each injection.
Bottom: Stimulation of mPOANts::ChR2 neurons increased the amount of time spent in the photostimulation side of the chamber and steroid priming resulted in an amplified increase in real-time preference for weeks following hormone treatment, compared to those treated with vehicle and mPOANts::eYFP controls (Two-Way ANOVA, interaction F10,75 = 8.91 p < 0.0001, n = 5 – 7 mice per group). E2: estradiol, Veh: vehicle.
b. Photostimulation increased the amount of time mPOANts::ChR2-eYFP males spent in the female zone (Two-Way ANOVA, Interaction F1,8 = 15.20, p = 0.0046, eYFP n= 6, ChR2 n = 5).
c. Photostimulation did not affect the amount of time mPOANts::ChR2-eYFP males spent in the male zone (Two-Way ANOVA, Interaction F1,8 = 5.21, p = 0.0564, eYFP n= 6, ChR2 n = 5).
d. Photostimulation enhanced female preference in mPOANts::ChR2-eYFP males (Two-Way ANOVA, Interaction F1,8 = 15.80, p = 0.0033, eYFP n= 6, ChR2 n = 5).
e. Mating experience in conjunction with steroid priming enhanced male preference (Two-Way ANOVA, Group F5,35 = 3.68, p = 0.0088, n = 7 mice per group).
f. Photoinhibition did not affect amount of time spent in the female zone (Two-Way ANOVA, Group F1,12 = 0.045, p = 0.8353, n = 7 mice per group).
g. Photoinhibition did not affect real-time place aversion in mPOANts::NpHR females or mPOANts::eYFP controls across vehicle or steroid-primed conditions (Two-Way ANOVA, interaction, F2,16 = 2.52, p = 0.1118, n = 7 mice per group).
h. Schematic illustrates chronic fiber placements in the mPOA from ChR2 and NpHR optogenetic behavioral cohorts and placements in the VTA from ChR2 mice.
Supplementary Figure 10 Optogenetic modulation of mPOANts neurons during a food assay.
a. Schematic illustrates two-chamber assay with a control zone (empty cup) and a food zone (high-fat diet cup). Light stimulation was delivered in alternating 10 min time epochs.
Color heat maps illustrating the spatial location in the food preference assay for a representative previously-primed mPOANts::eYFP control mouse.
Color heat maps illustrate the spatial location in the food preference assay for a representative previously-primed mPOANts::ChR2 mouse.
b. No group by time interaction was detected in the amount of time spent in the food zone mice (Two-Way ANOVA, interaction F4,26 = 1.04 p = 0.4044, n = 5-6 mice per group).
c. No group by time interaction was detected in the amount of time spent in the food zone mice (Two-Way ANOVA, interaction F4,26 = 0.21 p =.09322 n = 5-6 mice per group).
d. Optogenetic stimulation increased the number of food-zone approach bouts in previously-primed mPOANts::ChR2 females compared to vehicle-treated mPOANts::ChR2 and previously-primed eYFP control mice. (Two-Way ANOVA, interaction F4,26 = 6.51 p = 0.0009, n = 5-6 mice per group).
e. Optogenetic stimulation did not affect the number of control cup approach bouts and this did not differ by group. (Two-Way ANOVA, interaction F4,26 = 0.24 p = 0.9122, n = 5-6 mice per group).
f. No group differences of light stimulation were detected in time preference spent in the food zone over the control zone (One-Way ANOVA, interaction F2,13 = 0.44 p = 0.6531, n = 5-6 mice per group).
g. Optogenetic stimulation increased the rate per minute that previously-primed mPOANts::ChR2 females made direct contact with the food compared to vehicle-treated mPOANts::ChR2 and previously-primed eYFP control mice (Two-Way ANOVA, interaction F2,15 = 7.51 p = 0.0055, n = 5-6 mice per group).
h. Optogenetic stimulation did not affect the amount of time spent eating. (Two-Way ANOVA, interaction F4,26 = 2.14 p = 0.1050, n = 5-6 mice per group).
i. Optogenetic stimulation did not affect food consumption. (One-Way ANOVA, interaction F2,17 = 0.15 p = 0.8640, n = 6 mice per group).
j. Optogenetic inhibition did not affect the amount of time spent in the food zone. (Two-Way ANOVA ANOVA, interaction F2,22 = 0.47 p = 0.9543, n = 6-7 mice per group).
k. Optogenetic inhibition did not affect the amount of time spent in the control zone. (One-Way ANOVA, interaction F2,22 = 0.74 p = 0.4868, n = 6-7 mice per group).
l. Optogenetic inhibition did not affect preference for the food zone (One-Way ANOVA, interaction F1,11 = 3.82, p = 0.0766, n = 6-7 mice per group).
m. Optogenetic inhibition did not affect the number of visits into food zone. Two-Way ANOVA ANOVA, interaction F2,22 = 0.69 p = 0.5142, n = 6-7 mice per group).
n. Optogenetic inhibition did not affect the number of visits into the control zone. (Two-Way ANOVA, interaction F2,22 = 0.405 p = 0.6719, n = 6-7 mice per group).
o. Optogenetic inhibition did not affect the amount of high fat diet consumption. (un-paired t-test, t1,10 = 0.70 p = 0.4983, n = 6 mice).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–10 (PDF 4665 kb)
Basal in vivo Ca2+ dynamics in mPOANts neurons.
Representative video of spontaneous baseline Ca2+ dynamics in mPOANts::GCaMP6s neurons during in vivo two-photon imaging (2p) of an awake head-fixed female in proestrus. Imaging was acquired at 5 Hz but sped up ×4. (MP4 18928 kb)
Male odor–evoked Ca2+ dynamics in mPOANts neurons.
Representative video of Ca2+ dynamics in mPOANts::GCaMP6s neurons during in vivo two-photon imaging in an awake head-fixed female in proestrus. Imaging was acquired at 5 Hz but sped up ×4. Two concatenated odor trials are shown. Male odor was delivered at ~3–5 s and ~13–15 s. (MP4 4372 kb)
Optical self-stimulation of mPOANts neurons during proestrus.
Representative video of a mPOANts::ChR2 female in proestrus during optical self-stimulation test. In this video the female nose-pokes through an active port on the left to receive 20 Hz photostimulation with a 5 ms pulse width and for a duration of 3 s. (MP4 10924 kb)
Real-time place preference induced by mPOANts neuronal stimulation during proestrus.
Representative video of a mPOANts::ChR2 female in proestrus during a real-time place preference optogenetic behavioral assay. In this video then female received 20 Hz photostimulation only in the right side of the arena. (MP4 17107 kb)
Male preference induced by mPOANts neuronal stimulation.
Representative video of a mPOANts::ChR2 ovariectomized female that was previously steroid-primed. In this video the female is being photostimulated at 20 Hz during a social preference assay with two stimulus mice in the holding chambers: an adult male in the left chamber and an ovariectomized adult female on the right chamber. (MP4 15018 kb)
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McHenry, J., Otis, J., Rossi, M. et al. Hormonal gain control of a medial preoptic area social reward circuit. Nat Neurosci 20, 449–458 (2017). https://doi.org/10.1038/nn.4487
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DOI: https://doi.org/10.1038/nn.4487
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