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Synapse-specific astrocyte gating of amygdala-related behavior

Nature Neuroscience volume 20, pages 1540–1548 (2017)Cite this article

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Abstract

The amygdala plays key roles in fear and anxiety. Studies of the amygdala have largely focused on neuronal function and connectivity. Astrocytes functionally interact with neurons, but their role in the amygdala remains largely unknown. We show that astrocytes in the medial subdivision of the central amygdala (CeM) determine the synaptic and behavioral outputs of amygdala circuits. To investigate the role of astrocytes in amygdala-related behavior and identify the underlying synaptic mechanisms, we used exogenous or endogenous signaling to selectively activate CeM astrocytes. Astrocytes depressed excitatory synapses from basolateral amygdala via A1 adenosine receptor activation and enhanced inhibitory synapses from the lateral subdivision of the central amygdala via A2A receptor activation. Furthermore, astrocytic activation decreased the firing rate of CeM neurons and reduced fear expression in a fear-conditioning paradigm. Therefore, we conclude that astrocyte activity determines fear responses by selectively regulating specific synapses, which indicates that animal behavior results from the coordinated activity of neurons and astrocytes.

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Figure 1: Endogenously mobilized eCBs mediate CB1R-dependent increases in astrocytic calcium levels, enhance inhibitory synaptic transmission in CeL–CeM synapses and depress excitatory synaptic transmission in BLA–CeM synapses.
Figure 2: Astrocytic CB1R regulation of synaptic transmission relays on astrocytic calcium activity.
Figure 3: Selective expression and activation of astrocytic Gq-DREADDs in the CeM increases astrocytic calcium levels, increases inhibitory synaptic transmission at CeL–CeM synapses and depresses excitatory synaptic transmission at BLA–CeM synapses.
Figure 4: Selective activation of astrocytic DREADDs in CeM reduces the firing rate and decreases fear expression in a delayed fear conditioning paradigm.

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Acknowledgements

We thank J. Chen (UCSD, La Jolla, California, USA) for providing IP3R2− mice, and W. Buño, G. Perea, M. Navarrete and A. Covelo for helpful comments. This work was supported by NIH–NINDS (R01NS097312-01 to A.A.), the Human Frontier Science Program (Research Grant RGP0036/2014 to A.A. and G.M.), INSERM (to G.M.), Fondation pour la Recherche Medicale (DRM20101220445 to G.M.) and the China Scholarship Council (to Z.Z.). We thank the MnDRIVE Optogenetics Core at the University of Minnesota for technical support, and B. Roth and the UNC Vector Core (Chapel Hill, North Carolina, USA) for providing the Gq-DREADD adeno-associated virus.

Author information

Authors and Affiliations

  1. Department of Neuroscience, University of Minnesota, Minneapolis, Minnesota, USA

    Mario Martin-Fernandez, Stephanie Jamison & Alfonso Araque

  2. INSERM, U1215 NeuroCentre Magendie, Endocannabinoids and Neuroadaptation, Bordeaux, France

    Laurie M Robin, Zhe Zhao & Giovanni Marsicano

  3. Université de Bordeaux, Bordeaux, France

    Laurie M Robin, Zhe Zhao & Giovanni Marsicano

  4. Instituto Cajal, Consejo Superior de Investigaciones Científicas, Madrid, Spain

    Eduardo D Martin

  5. Hospital Nacional de Parapléjicos, Servicio de Salud de Castilla–La Mancha, Toledo, Spain

    Juan Aguilar

  6. Mouse Behavior Core, University of Minnesota, Minneapolis, Minnesota, USA

    Michael A Benneyworth

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  1. Mario Martin-Fernandez
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  2. Stephanie Jamison
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  3. Laurie M Robin
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Contributions

M.M.-F. and A.A. conceived the study. M.M.-F., J.A., G.M. and A.A. wrote the manuscript. M.M.-F. performed and analyzed electrophysiological and calcium imaging experiments and analyzed the data. M.M.-F., E.D.M., J.A. and A.A. designed in vivo electrophysiological experiments. S.J. performed immunohistochemistry techniques. M.M.-F., M.A.B., G.M. and A.A. designed the behavioral experiments. M.A.B., L.M.R. and Z.Z. performed behavioral experiments. All the authors read and edited the manuscript.

Corresponding author

Correspondence to Alfonso Araque.

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Integrated supplementary information

Supplementary Figure 1 Amygdaloid complex and evoked synaptic currents recorded in CeM amygdala neurons.

(a) Left: Schematic representation (modified from: Allen Brain Atlas) of coronal sections containing the amygdala subnuclei inside the square and antero/posterior coordinates from bregma in mm. CeM, represented in green, CeL in blue and BLA in red. Right: Schematic representation of amygdala subnuclei, cellular types and connectivity (b) Top: Images showing neuronal recordings from CeM (green) and stimulating electrodes in BLA (red) or CeL (blue). Scale bar, 500 μm Bottom: BLA-evoked glutamatergic EPSCs (bottom, left; isolated in the presence of GABAAR and GABABR antagonists picrotoxin and CGP) and CeL-evoked GABAergic IPSCs (bottom, right; isolated in the presence of glutamate receptor AMPAR and NMDAR antagonists CNQX and AP5). Scale bars 8 pA and 25 ms. (c) Image showing SR101 staining CeM astrocytes and blood vessels (arrow). Scale bars: top image 90 μm and bottom image 30 μm.

Supplementary Figure 2 Synaptic potency of CeL-evoked IPSCs and BLA-evoked EPSCs recorded in the heteroneuron after homoneuron ND in different experimental conditions.

(a) Synaptic potency of CeL-evoked IPSCs before and after homoneuron ND (time=0, n=22). (b) Synaptic potency of CeL-evoked IPSCs before and after homoneuron ND in control conditions (n=22, p=0.88), in the presence of AM251 (n=11, p=0.087), in GFAP-CB1RWT (n=7, p=0.12), GFAP-CB1R-/- (n=7, p=0.56) and IP3R2-/- (n=7, p=0.92) mice, in the presence of MPEP+LY (n=10, p=0.42), SCH (n=7, p=0.37) and CPT (n=11, p=0.82). (c) Synaptic potency of BLA-evoked EPSCs before and after homoneuron ND (time=0; n=24). (d) Synaptic potency of BLA-evoked EPSCs before and after homoneuron ND in control conditions (n=24, p=0.2), in the presence of AM251 (n=9, p=0.089), and in GFAP-CB1RWT (n=8, p=0.16), GFAP-CB1R-/- (n=10, p=0.24) and IP3R2-/- (n=10, p=0.21) mice, in the presence of MPEP+LY (n=13, p=0.13), CPT (n=9, p=0.75) and SCH (n=12, p=0.56). *P < 0.05, **P < 0.01, ***P < 0.001; Student’s paired t-test. Error bars indicate SEM.

Supplementary Figure 3 Basal probability of release of CeL-evoked IPSCs and BLA-evoked EPSCs.

(a) Basal probability of release of CeL-evoked IPSCs in control conditions (n=22), in the presence of AM251 (n=11), in GFAP-CB1RWT (n=7), GFAP-CB1R-/- (n=7) and IP3R2-/- (n=10) mice, in the presence of MPEP+LY (n=10), SCH (n=7) and CPT (n=13). No differences were found between the different experimental conditions (One-Way ANOVA, F7, 79 =1.96, p=0.071). (b) Basal probability of release BLA-evoked EPSCs in control conditions (n=24), in the presence of AM251 (n=12), and in GFAP-CB1RWT (n=11), GFAP-CB1R-/- (n=9) and IP3R2-/- (n=10) mice, in the presence of MPEP+LY (n=13), SCH (n=12) and CPT (n=9). No differences were found between the different experimental conditions (One-Way ANOVA, F7, 91 =1.93, p=0.073). Box-whisker plots indicate median, interquartile range and 10th-90th percentiles of the distribution.

Supplementary Figure 4 Differential astrocyte-mediated regulation of BLA-evoked EPSCs and CeL-evoked IPSCs in the same CeM neurons.

(a) DIC images and schemes showing neuronal recordings in CeM (green) and stimulating electrodes in BLA (red) or CeL (blue). Scale bar 500 μm. (b) IPSCs and EPSCs in basal conditions and after ND. IPSCs were evoked by stimulating the CeL and were isolated in the presence of CNQX and AP5; after the washout of CNQX and AP5, EPSCs evoked by stimulation of BLA were isolated with picrotoxin and CGP. Scale bars, 25 ms and 8 pA. (c) Left: CeL-evoked IPSCs Pr (blue) and BLA-evoked EPSCs Pr (red) before and after homoneuron ND (at time=0 and 50, respectively). Right: CeL-evoked IPSCs Pr before and after ND (n=6, p=0.02) and BLA-evoked EPSCs Pr before and after ND (n=6, p=0.04). (d) Left: Synaptic potency of CeL-evoked IPSCs (blue) and synaptic potency of BLA-evoked EPSCs (red) before and after homoneuron ND (at time=0 and 50, respectively). Right: Synaptic potency of CeL-evoked IPSCs before and after ND (n=6, p=0.1) and synaptic potency of BLA-evoked EPSCs before and after ND (n=6, p=0.96). *P < 0.05, **P < 0.01, ***P < 0.001; Student’s paired t-test. Error bars indicate SEM.

Supplementary Figure 5 Synaptic potency of CeL-evoked IPSCs and BLA-evoked EPSCs after CNO application in different experimental conditions.

(a) Left: Synaptic potency of CeL-evoked IPSCs before and after CNO pressure pulse application (time=0; n=7). Right: Synaptic potency of BLA-evoked EPSCs before and after CNO pressure pulse application (time=0; n=8). (b) Synaptic potency of CeL-evoked IPSCs and BLA-evoked EPSCs before and after CNO pressure pulse application in control (IPSCs n=6, p=0.93; EPSCs n=8, p=0.48), in AM251 (IPSCs n=8, p=0.87; EPSCs n=6, p=0.77) and in the presence of SCH (n=7, p=0.66) and CPT (n=7, p=0.12). (c) Left: Synaptic potency of CeL-evoked IPSCs before and after CNO bath application (time=0, n=9) and SCH application (time=30). Right: Synaptic potency of BLA-evoked EPSCs before and after CNO bath application (time=0; n=6) and CPT application (time=30). (d) Synaptic potency of CeL-evoked IPSCs in control conditions and after CNO bath application (n=8, p=0.3) and SCH bath application (n=3, p=0.91) and synaptic potency of BLA-evoked EPSCs before and after CNO bath application (n=6, p=0.59) and CPT bath application (n=4, p=0.24). *P < 0.05, **P < 0.01, ***P < 0.001; Student’s paired t-test. Error bars indicate SEM.

Supplementary Figure 6 Sustained activation of CeM astrocytes by bath application of CNO.

(a) Left: Fluorescence image showing mCherry and fluo4 pseudocolor images representing fluorescence intensities in CeM astrocytes before and after CNO bath application. Scale bar, 10μm. Right: Astrocytic calcium levels before and after CNO bath application. Scale bars, 45% and 60s. (b) Left: Time course of the calcium event frequency in basal condition and during CNO (at time=0). Right: Calcium event frequency in basal condition and during CNO (n=6, p=0.009) (c) Schematic representation and CeL-evoked IPSCs recorded in CeM neurons before and after CNO application. Scale bars, 15 pA and 25 ms. (d) Left: Time course of CeL-evoked IPSCs Pr before and after CNO application (time 0) and SCH application (time=30). Right: CeL-evoked IPSCs Pr before and after CNO application (n=9, p=0.002) and SCH application (n=3, p=0.88). (e) Schematic representation and BLA-evoked EPSCs recorded in CeM neurons before and after CNO application. Scale bars, 15 pA and 25 ms. (f) Left: Time course of BLA-evoked EPSCs Pr before and after CNO application (time 0) and CPT application (time=30). Right: BLA-evoked EPSCs Pr before and after CNO application (n=6, p=0.0001) and CPT application (n=4, p=0.23). *P < 0.05, **P < 0.01, ***P < 0.001; Student’s paired t-test. Error bars indicate SEM.

Supplementary Figure 7 CNO effects in mice lacking DREADDs expression.

(a) Left: Fear response measured as percentage of freezing during the 15 sec cue presentation in sham-surgeries mice during fear conditioning, Right: Fear response measured as percentage of freezing during the 3 minutes of continuous CS presentation, data depicted in 1-minute time bins. One non-reinforced CS was presented in Test 1 and in Test 2 (n=10 mice injected with CNO, red, and 10 mice injected with saline, grey). No differences were observed between saline and CNO injected mice in any of the three time bins of Test 1 (p=0.88, p=0.39 and p=0.24) and Test 2 (p=0.99, p=0.81 and p=0.7). (b) Percentage of the time spent in the open arms in the elevated plus maze test (n=10 mice injected with CNO and 9 mice injected with saline). No differences were observed between saline and CNO injected mice (p=0.24). *P < 0.05, **P < 0.01, ***P < 0.001; Unpaired Student’s t-test. Box-whisker plots indicate median, interquartile range and 10th-90th percentiles of the distribution.

Supplementary Figure 8 Schematic of the proposed model consistent with present findings.

(a) The schematic shows that either an endogenous stimulus (eCBs; Figure 1 and 2) or a selective exogenous stimulus (DREADDs activation by CNO; Figure 3) are able to activate astrocytes and induce a regulation in CeL-CeM and BLA-CeM synapses. The astrocytic activity increases synaptic probability of release in CeL-CeM synapses through activation of A2A receptors and decreases synaptic probability of release through activation of A1 receptors in BLA-CeM synapses (Figures 1 and 3). (b) The main amygdala subnuclei are represented: the basolateral amygdala (BLA; red), the lateral portion of the central amygdala (CeL, Blue) and the medial portion of the central amygdala (CeM, green). GABAergic neurons are represented as blue circles and glutamatergic neurons as red circles. Consistently with the synaptic effects, upon astrocytic activation the firing rate of CeM neurons and the fear expression are decreased (Figure 4).

Supplementary Figure 9 GFAP-CB1R-/- mice present impairments in astrocytic CB1R effects but not in the neuronal CB1R dependent depolarization suppression of inhibition (DSI).

(a) Left: Astrocytic calcium levels before and after ND (black) in the GFAP-CB1R-/- mice. Scale bars 20 s and 50 %. Right: Calcium event probability before and after ND at time=0 in GFAP-CB1RWT (n=10) and in GFAP-CB1R-/- (n=9) mice. (b) Left: Homoneuronal CeL-evoked IPSCs Pr before and after homoneuron ND (at time=0; n=6) in GFAP-CB1R-/- mice. Right: Homoneuronal CeL-evoked IPSCs Pr before and after homoneuron ND in in GFAP-CB1RWT (n=6, p=0.015) and GFAP-CB1R-/- (n=6, p=0.014). *P < 0.05, **P < 0.01, ***P < 0.001; paired Student’s t-test. Error bars indicate SEM.

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Martin-Fernandez, M., Jamison, S., Robin, L. et al. Synapse-specific astrocyte gating of amygdala-related behavior. Nat Neurosci 20, 1540–1548 (2017). https://doi.org/10.1038/nn.4649

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