Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.
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Python code (used for ex vivo calcium imaging data analysis) can be found in Supplementary Software. All data that support the findings of this study are available from the corresponding authors upon reasonable request.
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This work was supported by the Centre National de la Recherche Scientifique contract UPR3212, the Université de Strasbourg contract UPR3212, IASP Early Career Research grant 2012, FP7 Career Integration grant 334455, Initiative of Excellence (IDEX) Attractiveness grant 2013, IDEX Interdisciplinary grant 2015, University of Strasbourg Institute for Advanced Study (USIAS) fellowship 2014-15, Foundation Fyssen research grant 2015, NARSAD Young Investigator grant 24821, ANR JCJC grant 19-CE16-0011-0 (to A.C.), ANR-DFG grant GR 3619/701 (to A.C. and V.G.), Alexander von Humboldt fellowship (to D.H.), Seed grant from DFG within the Collaborative Center SFB 1158 and Fyssen Foundation fellowship (to A.L.); DFG grants GR 3619/13-1, GR 3619/15-1 and GR 3619/16-1 (to V.G.); SFB 1158 (to C.P., J.S. and V.G.), SNSF-DFG grant GR 3619/8-1 (to R.S. and V.G.), the Fritz Thyssen Foundation (to V.G.), DFG Postdoctoral Fellowship AL 2466/1-1 (to F.A.), the Alexander von Humboldt Foundation (to D.H.), the Fyssen Foundation and PROCOP grant and SFB1158 seed grant for young scientists (to A.L.), post-doctoral fellowship from the Fund for Scientific Research Flanders (12V7519N) (to J.W.), Russian Science Foundation RSF (17-75-10061) and the subsidy allocated to the Kazan Federal University for the State Assignment, no. 0671-2020-0059 (to A.R.), the intramural research program of the NIMH (ZIAMH002498) (to W.S.Y.), National Institutes of Health grants R01NS094640 and R01HL090948, SFB/TRR 152 and ERC-CoG-772395 (to J.E.S.), the European Research Council (Consolidator Grant no. 683154) and the European Union’s Horizon 2020 Research and Innovation Program (Marie Sklodowska-Curie Innovative Training Networks, grant no. 722053, EU-GliaPhD) (to N.R.). The authors thank V. Lelièvre for in situ hybridization advice; R. Goutagny for in vivo optogenetics assistance; F. Magara for anxiety behavior advice; B. Kurpiers and the Interdisciplinary Neurobehavioral Core Facility of Heidelberg University for experiments performed there; S. Reibel and the Chronobiotron UMS 3415 for all animal care; and T. Splettstoesser (www.scistyle.com) for help with the preparation of Fig. 7.
The authors declare no competing interests.
Peer review information Nature Neuroscience thanks Thomas Papouin, Gertrudis Perea, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Validation of OTR expression in CeL astrocytes using several astrocytic markers.
a, Combination of FISH and IHC showing co-localization of GS (top left), NeuN (bottom left) and both markers (right) with OTR mRNA in rats. Scale bars are 100µm and 10µm. (n rats = 5, n slices = 20, n astrocytes = 1354, n neurons = 1254). b, proportion of OTR positive astrocytes and neurons in mice. n astrocytes = 897; n neurons = 688; n rats = 4 c, (top left) FISH overview for OTR mRNA (green), counterstained with polyclonal anti-ALDH1L1 antibody (red) in CeA. (bottom left) High magnification image of cells positive for both OTR mRNA and ALDH1L1 (double arrows); green arrows point OTR mRNA-positive cells; red arrows point ALDH1L1-positive cells. Scale bars: 400 (Top) and 50µm (Bottom). (Right) Quantification of ALDH1L1-positive cells positive for OTR mRNA. n astrocytes = 450; n rats = 4. d, RNAscope in situ hybridization showing GFAP (red) and OTR (green) expressing cells in mice CeA. Merged images include DAPI stain (blue); n = 3. (Bottom) Negative control probe targeting the bacterial gene DapB; n = 3. Scale bar is 10μm. st: stria terminalis. Data are expressed as mean across slices plus SEM and white circles represent individual cell data. (Statistics and numbers in Supplementary Table 1).
a, (left) C1V1-mCherry (OxyOpto) expressing oxytocin neuron of the PVN. Scale bar is 20µm; n = 11. (right) Schematic representation of the whole cell patch clamp of the OxytOpto-expressing cells of the PVN. b, (left) λ542nm light exposure (yellow line) induce a depolarizing current, (right) enabling precise spiking of OxytOpto-expressing cells. c, Typical confocal image of CeL astrocytes co-labeled with SR101 and OGB1; n = 46. Scale bar 20μm. d, (left) Pseudo-color pictures of an SR101 positive cell identified as an astrocyte compared to neurons identified under oblique infrared light, Scale bar 10μm. (Middle) Electrophysiological properties of patched SR101+ (red, n = 82) and SR101- cells (grey, n = 20). Medians, quartiles and whiskers can be found in Supplementary Table 2. (Right) Typical responses to 20pA current steps of a SR101+ (red) and a SR101- cells (black). e, CeL SR101 positive cell filled with biocytin through whole cell patch-clamp (green) lacks NeuN signal (Blue); n = 3. Scale bar 50 μm. f, Illustration of calcium imaging data analysis method. (left) Fluorescence traces were splited into a “baseline” and a “post-stimulation” trace. Calcium transients were detected according to a prominence threshold and their frequency was quantified before and after the drug application. All data were averaged across astrocytes per slice, and this results was used as statistical unit. All data were compared (before vs. after drug application) and the results were expressed in ratio (baseline/drug effect), a ratio of 1 meaning neither an increase nor a decrease of the measured parameter. ns = 7, na = 36. g, (top left) Immunohistochemical staining for glutamine synthase (GS; blue), Cre-GFP (green) and Cre recombinase (red). (bottom left) Overview of the CeA, displaying correct viral targeting of the CeL subdivision. (right) Quantification of efficiency and specificity of the transduction. Efficiency: Over 1001 GS positive cells, 561 were also GFP positive, indicating an efficiency of 56±4.9%. Specificity: we counted a total of n = 977 GFP-positive cells, 940 of which were positive for GS (96.2±2.1%). None of the GFP or Cre signals were detected in NeuN positive cells (0 out of n = 850, 4 mice, not shown). Finally, 99.82 ± 0.2% of GS-positive astrocytes containing GFP signal were Cre-positive (n = 1001). n mice = 4. Scale bars: 400 (Bottom) and 20µm (Top). h, Immunohistochemical analysis of OTR expression in NeuN-positive cells of the CeL revealed no difference in OTR levels between control and GFAP-Cre injected animals; 85.0 ± 1.6% (n = 688, n = 4 mice) and 86.7 ± 1.6% (n = 660, n = 4 mice) respectively. Scale bars: 20µm. Data in f-h are expressed as mean across slices plus SEM and white circles represent individual cell data. ***p < 0.001, two-sided Mann-Whitney U test. (Statistics and numbers in Supplementary Table 2).
Extended Data Fig. 3 Astro-astrocytic communication in the CeL do not depends on purinergic nor NMDAR-dependent signalling.
a, Illustration of the 3D reconstruction and the quantification of the number of contact between astrocytes. (left) Pictures of the raw fluorescence obtained after anti-GFAP immunohistochemistry, (middle) based on this staining, we performed a 3D reconstruction of astrocytes and (right) evaluate the number of astrocyte-astrocyte contact using the given formula. With n = upper limit of summation, i = index of summation, xi = typical element (that is astrocytic contacts with 1µm or less proximity). With this approach, we quantified all astrocyte contacts without discrimination between individual astrocyte entities. b, (left) Typical ΔF traces following TGOT+TTX application in presence of PPADS (50 µM) + Suramin (75 µM) + A438079 (1 µM). (right) Proportion of responding astrocytes, AUC of ΔF traces and Ca2+ transients frequency normalized to baseline values following application of TGOT+TTX (0.4 μM) in presence of PPADS (50 µM) + Suramin (75 µM) + A438079 (1 µM) (ns = 10, na = 112). c, (left) Typical ΔF traces following TGOT+TTX application in presence of CPT (5 µM) or SCH 58261 (100 nM). (right) Proportion of responding astrocytes, AUC of ΔF traces and Ca2+ transients frequency normalized to baseline values following application of TGOT+TTX (0.4 μM) in presence of CPT (ns = 7, na = 22) or SCH (ns = 8, na = 32). d, (left) Typical ΔF traces following TGOT+TTX application in presence of AP5 (50 µM). (right) Proportion of responding astrocytes, AUC of ΔF traces and Ca2+ transients frequency normalized to baseline values following application of TGOT+TTX (0.4 μM) in presence of AP5 (ns = 6, na = 55). Data are expressed as mean across slices plus SEM and white circles represent individual cell data. White circles indicate average across astrocytes per slice. (Statistics and numbers in Supplementary Table 3).
Extended Data Fig. 4 Specific optogenetic stimulation of CeL astrocytes elicit calcium transients in a Ca2+-dependent manner.
a, (left) Immunohistochemistry image shows CeL cells transfected with rAAV-Gfap-C1V1(t/t)-mCherry (AstrOpto) with co-labeling for ALDH1L1. White arrow shows one cell expanded in insets. Scale bars are 25 and 10 µm (insets). (right) Quantification of the efficiency and specificity of transduction of C1V1 in CeL astrocytes. PGFAP- C1V1-mCherry rAAV vector was injected into rat CeA (bilaterally, 200nl). Specificity: Over 1090 mCherry-positive cells, 98.8±0.7% were positive for ALDH1L1. None of the analyzed cells were positive for NeuN. Efficiency: Over 1090 ALDH1L1 positive cells, 62.3±3.5% were also mCherry positive. n = 4 rats. b, (left) Typical ΔF traces following AstrOpto activation. λ542nm (1s @ 0.5Hz during 3 minutes) with (top 3 traces) or without (bottom 3 traces) extracellular calcium. (right) Proportion of responding astrocytes, AUC of ΔF traces and Ca2+ transients frequency normalized to baseline values following AstrOpto stimulation with (ns = 12, na = 49) or without Ca2+ in the ACSF (ns = 12, na = 53). Data are expressed as mean across slices plus SEM. White circles indicate average across astrocytes per slice. **p<0.01, ***p<0.001, two-sided Mann-Whitney U test. (Statistics and numbers in Supplementary Table 4).
Figure 5. a, DAAO (0.15 IU/ml, incubation time > 1h30; n = 7) and AP5 (50 µM; n = 6) prevent the effect of photoactivation of astrocytes (AstrOpto) on IPSC frequency in CeM neurons. b, Two consecutive TGOT application effects on IPSCs frequencies in CeM neurons after DAAO (0.15 U/ml, incubation time > 1h30) followed by D-Serine (20 min, 100 μM) incubation of the same cells (n = 7). c, dOVT (1 µM; n = 6) prevents the effect of TGOT on IPSCs frequencies in CeM neurons. d, Effect of double (20 min apart) application of TGOT on IPSCs frequencies in CeM (0.4 µM n = 7). e, Effect of DCKA (10 µM, n = 15) and DNQX (25 µM; n = 10) on TGOT-induced increase in IPSC frequency in CeM neurons. f, Purinergic antagonists do not prevent TGOT effect on IPSC frequency in CeM neurons. PPADS (50 µM; n = 9), CPT (5 µM; n = 11), SCH (100 nM; n = 10). Data are expressed as averages plus SEM and white circles represent individual cell data. #p < 0.05, ##p < 0.01, ###p < 0.001, Friedman and Dunn’s Multiple comparisons, **p < 0.01, ***p < 0.001, two-sided unpaired t-test or Mann-Whitney U test. (Statistics and numbers in Supplementary Table 5).
Extended Data Fig. 6 SNI procedure does not modify the effect of OTR activation on CeL microcircuit activity.
a, 30 days post surgeries time course of mechanical pain threshold evolution across sham (n = 23) and SNI (n = 22) rats. Data are expressed as mean across animals plus SEM. b, (left) Typical ΔF traces following TGOT+TTX application in SNI rats. (right) Proportion of responding astrocytes, AUC of ΔF traces and Ca2+ transients frequency normalized to baseline values following application of TGOT+TTX (0.4 μM) in sham (ns = 16, na = 74) or SNI (ns = 18 et na = 136) rats. Data are expressed as mean across slices plus SEM. White circles indicate average across astrocytes per slice. c, TGOT effect on CeM neurons IPSCs frequencies is unchanged between Sham (n = 9) or SNI (n = 9) rats. Data are expressed as average plus SEM and white circles represent individual cell data. d, Two consecutive TGOT application effects on IPSCs frequencies in CeM neurons after DAAO (0.15 U/ml, incubation time > 1h30) followed by D-Serine (20 min, 100 μM) incubation of the same cells from acute brain slices of SNI animals (n = 11). Data are expressed as averages plus SEM and white circles represent individual cell data. e, Verification of cannulae implantation sites. (left) Schematic representation of the stereotaxic injection viewed in a coronal slice; n rats = 67, n mice = 52. (middle, right) Infrared and fluorescence microscopy picture showing the injection site of fluorescent latex beads in the CeL on horizontal slices. Scale is 1mm. ** p<0.01, *** P<0.001, two-sided paired t-test or Wilcoxon test, #p<0.05 Friedman test followed by Dunn’s multiple comparison. (Statistics and numbers in Supplementary Table 6).
Extended Data Fig. 7 Effects of CeL astrocytes and OTR activities manipulations on locomotion and contralateral hindpaw sensitivity.
a,e, Experimental strategy for the specific expression of C1V1 in mice CeL astrocytes (a, AstrOpto) or the specific deletion of OTRs in mice CeL astrocytes (e, GFAP OTR KO). The treatments applied are color coded as the legend key indicate. Control indicate a vehicle injection. b,f, Mechanical pain threshold was assessed on the non-injured paw of SNI (bottom, gray box) and its equivalent in Sham (top) groups. TGOT or its vehicle, or astrocytes light-evoked activation of C1V1, were administered in the CeL and mechanical pain threshold assessed again at different time points. Rats (n): Sham control n = 12, TGOT n = 10, C1V1 n = 6; SNI control n = 12, TGOT n = 10, C1V1, n = 9. Mice (n): Sham WT n = 6, OTR cKO n = 8; SNI WT n = 5, OTR cKO n = 13. c,g, Locomotion was assessed through measurement of the distance travelled during the length of the elevated plus maze experiment, after administration of the different treatments. Rats (n): Sham control n = 10, TGOT n = 8, C1V1 n = 9; SNI control n = 20, TGOT n = 9, C1V1, n = 7. Mice (n): Sham WT Veh n = 7, WT TGOT n = 6; Sham OTR cKO Veh n = 5, OTR cKO TGOT n = 7; SNI WT Veh n = 7, WT TGOT n = 8; SNI OTR cKO Veh n = 5, OTR cKO TGOT n = 7. d,h, Locomotion was assessed through measurement of the distance travelled during the time of the conditioned place preference experiment, after administration of the different treatments. Rats (n): Sham control n = 13, TGOT n = 6, C1V1 n = 5; SNI control n = 10, TGOT n = 8, C1V1, n = 8. Mice (n): Sham WT Veh n = 5, WT TGOT n = 8; Sham OTR cKO Veh n = 5, OTR cKO TGOT n = 4; SNI WT Veh n = 5, WT TGOT n = 5; SNI OTR cKO Veh n = 4, OTR cKO TGOT n = 6. Data are expressed as mean across animals plus SEM. (Animals number and Statistics in Supplementary Table 7).
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Wahis, J., Baudon, A., Althammer, F. et al. Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents. Nat Neurosci 24, 529–541 (2021). https://doi.org/10.1038/s41593-021-00800-0
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