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Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour


Astrocytes associate with synapses throughout the brain and express receptors for neurotransmitters that can increase intracellular calcium (Ca2+)1,2,3. Astrocytic Ca2+ signalling has been proposed to modulate neural circuit activity4, but the pathways that regulate these events are poorly defined and in vivo evidence linking changes in astrocyte Ca2+ levels to alterations in neurotransmission or behaviour is limited. Here we show that Drosophila astrocytes exhibit activity-regulated Ca2+ signalling in vivo. Tyramine and octopamine released from neurons expressing tyrosine decarboxylase 2 (Tdc2) signal directly to astrocytes to stimulate Ca2+ increases through the octopamine/tyramine receptor (Oct-TyrR) and the transient receptor potential (TRP) channel Water witch (Wtrw), and astrocytes in turn modulate downstream dopaminergic neurons. Application of tyramine or octopamine to live preparations silenced dopaminergic neurons and this inhibition required astrocytic Oct-TyrR and Wtrw. Increasing astrocyte Ca2+ signalling was sufficient to silence dopaminergic neuron activity, which was mediated by astrocyte endocytic function and adenosine receptors. Selective disruption of Oct-TyrR or Wtrw expression in astrocytes blocked astrocytic Ca2+ signalling and profoundly altered olfactory-driven chemotaxis and touch-induced startle responses. Our work identifies Oct-TyrR and Wtrw as key components of the astrocytic Ca2+ signalling machinery, provides direct evidence that octopamine- and tyramine-based neuromodulation can be mediated by astrocytes, and demonstrates that astrocytes are essential for multiple sensory-driven behaviours in Drosophila.

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Figure 1: Larval chemotaxis and startle-induced responses require the astrocyte-expressed TRP channel Wtrw.
Figure 2: Tdc2+ neurons fire rhythmically to drive astrocyte somatic Ca2+ transients through the Oct-TyrR.
Figure 3: Astrocytes mediate Tyr-induced inhibition of dopaminergic neurons through Oct-TyrR.
Figure 4: Tyr-mediated inhibition of dopaminergic neurons depends on adenosine receptors and glial endocytic function.


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We thank our colleagues, the Vienna Drosophila RNAi Center and the Bloomington Stock Center for providing fly stocks, members of the Freeman laboratory for comments on the manuscript and A. Sheehan for generating the wtrw::gfp construct. This work was supported by NINDS grant R01 NS053538 (to M.R.F.). During the period of this study M.R.F. was an Investigator with the Howard Hughes Medical Institute.

Author information

Authors and Affiliations



Z.M. and M.R.F. designed experiments. Z.M. performed all experiments. T.S. provided alrm-LexA::GAD transgenic flies. D.E.B. provided unpublished data on norepinephrine-mediated activation of mammalian astrocytes and input that helped guide the course of the study. Z.M. and M.R.F. wrote the manuscript with editing by T.S.

Corresponding author

Correspondence to Marc R. Freeman.

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The authors declare no competing financial interests.

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Reviewer Information

Nature thanks L. Luo, B. MacVicar, L. Vosshall and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Synchronous somatic Ca2+ transients in Drosophila astrocytes.

ac, Brief diagrams of behavioural tests. d, tsh-Gal80 suppression of alrm-Gal4 activity. Nc82, neuropil (NP). Astrocytes (Astro), alrm > myr::tdTomato. Vnc, ventral nerve cord. Scale bar, 50 μm. e, f, Chemotaxis assay (n = 12). g, Locomotion assay (n listed). h, Light avoidance assay (n = 12). i, Gentle touch assay (n = 24). j, GCaMP6s and mCherry expression in astrocytes. Scale bar, 50 μm. k, Representative pseudocoloured images of four continuous Ca2+ transients. Scale bar, 50 μm. l, Traces of normalized GCaMP6s intensity over mCherry of 10 individual astrocytes in 15-min live imaging windows. m, Averaged traces of individual somatic Ca2+ transients. n, Somatic Ca2+ transients in astrocytes treated with TTX and LaCl3 (n = 10, 160 cells). o, GCaMP6s expression in astrocytes and traces of 10 individual astrocytes from an intact larva. Scale bar, 20 μm. Grey bars (l, o) represent population rise or fall in GCaMP6s signals. p, GCaMP6s-labelled astrocytes and R-GECO1-labelled Tdc2+ neurites and their averaged traces in an intact larva. Scale bar, 20 μm. *P < 0.05, **P < 0.01; NS, not significant; error bars, s.e.m. Wilcoxon and Mann–Whitney tests followed by Bonferroni-Holm post hoc test (e, n), one-way ANOVA followed by Tukey’s post hoc test (fi).

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Extended Data Figure 2 Somatic Ca2+ transients in astrocytes inhibit the activity of dopaminergic neurons.

ac, Representative traces of astrocyte Ca2+ transients with blockade of Tyr and Oct signalling. d, Stimulation of olfactory neurons activates Tdc2+ neurons (n = 3–4). Scale bar, 25 μm. e, Activity of Tdc2+ neurons is not altered in wtrw mutants (n = 8, 48 neurites). f, Astrocytes, Tdc2+ neurons and dopaminergic neurons in larval CNS. Dorsal (arrows point to astrocyte somas labelled with anti-GAT antibody), medial (neurites intermingled with ramified processes of astrocytes for monitoring activity are labelled) and ventral (cell bodies of tdc2>myr::tdTomato, Gal4/UAS and th>GCaMP6s LexA/LexAop dopaminergic neurons) images from the boxed region are shown (right). s, subesophageal. t, thoracic. Scale bar, 50 μm. g, Amplitude of Ca2+ spikes in dopaminergic neurons (n = 10, 80 neurites). h, i, Chemotaxis assay (n listed). j, Number of Ca2+ spikes of dopaminergic neurons (n = 6, 48 neurites). k, Responses of astrocytes to Tyr (0.5 mM) in the presence of TTX (n = 6, 96 cells total). l, Number of Ca2+ spikes in dopaminergic neurons (n = 6, 48 neurites). m, AITC induces Ca2+ influx into astrocytes expressing TrpA1 (n = 5, 80 cells). Scale bar, 50 μm. n, Number of Ca2+ spikes in dopaminergic neurons (n = 6, 48 neurites). *P < 0.05, **P < 0.01; NS, not significant; error bars, s.e.m. One-way ANOVA followed by Tukey’s post hoc test.

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Supplementary information

Synchronous somatic calcium transients in Drosophila astrocytes

Top left: mCherry. Top right: GCaMP6s. Bottom left: merge. Video was sped up X100. (WMV 1109 kb)

Concomitant activity of Tdc2+ neurons and astrocytes

Top left: GCaMP6s labeled astrocytes. Top right: R-GECO1 labeled Tdc2+ neurites. Bottom left: merge. Video was sped up X50. (WMV 12002 kb)

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Ma, Z., Stork, T., Bergles, D. et al. Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour. Nature 539, 428–432 (2016).

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