Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour

Abstract

Astrocytes associate with synapses throughout the brain and express receptors for neurotransmitters that can increase intracellular calcium (Ca²⁺). Astrocytic Ca²⁺ signalling has been proposed to modulate neural circuit activity, 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.

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

a-c, Brief diagrams of behavioral tests. d, tsh-Gal80 suppression of alrm-Gal4 activity. Nc82, neuropil (NP). Astrocytes (Astro), alrm>myr::mtdTomato. Vnc, ventral nerve cord. Scale bar, 50μm. 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, 50μm. k, Representative pseudocolored images of 4 continuous Ca²⁺ transients. Scale bar, 50μm. l, Traces of normalized GCaMP6s intensity over mCherry of 10 individual astrocytes in 15min live imaging window. m, Averaged traces of individual somatic Ca²⁺ transients. n, somatic Ca²⁺ transients in astrocytes with treatments of TTX and LaCl₃ (n=10, 160 cells). o, GCaMP6s expression in astrocytes and traces of 10 individual astrocytes from an intact larva. Scale bar, 20μm. Grey bars (l,o) represent population rise/fall in GCaMP6s signals. p, GCaMP6s labeled astrocytes and R-GECO1 labeled Tdc2⁺ neurites and their averaged traces in an intact larva. Scale bar, 20μm. *p<0.05, **p<0.01, n.s., not significant. Error bar, 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 (f,g,h,i).

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

a-c, Representative traces of astrocyte Ca²⁺ transients with blockade of tyramine/octopamine signaling. d, Stimulation of olfactory neurons activates Tdc2⁺ neurons (n=3-4). Scale bar, 25μm. e, Activity of Tdc2⁺ neurons are 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), medial (neurites intermingled with ramified processes of astrocytes for monitoring activity are labeled) and ventral (cell bodies of Tdc2⁺ and dopaminergic neurons) images from the boxed region are shown right. s, subesophageal. t, thoracic. Scale bar, 50μm. g, Amplitude of Ca²⁺ spikes of 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 tyramine (0.5mM) in the presence of TTX (n=6, 96 cells total). l, Number of Ca²⁺ spikes of dopaminergic neurons (n=6, 48 neurites). m, AITC induces Ca²⁺ influx to astrocytes expressing TrpA1 (n=5, 80 cells). Scale bar, 50μm. n, Number of Ca²⁺ spikes of dopaminergic neurons (n=6, 48 neurites). *p<0.05, **p<0.01, n.s., not significant. Error bar, s.e.m. One-way ANOVA followed by Tukey's post hoc test.

Figure 1. Larval chemotaxis and startle-induced responses require the astrocyte-expressed TRP channel Water witch

a, Chemotaxis assay (n=12). b, Gentle touch assay (n=30). c, Pseudocolored maximum intensity projections of 15min movies, averaged traces of 16 individual astrocytes and quantifications of the frequency of somatic Ca²⁺ transients (n=10, 160 cells total). Scale bar, 50μm. d, Responses of astrocytes to neurotransmitters/neuromodulators in the presence of tetrodotoxin (n=6, 96 cells total). *p<0.05, **p<0.01, n.s., not significant, Error bars, s.e.m. Wilcoxon and Mann-Whitney tests followed by Bonferroni-Holm post hoc test (a). One-way ANOVA followed by Tukey's post hoc test (b,c,d).

Figure 2. Tdc2⁺ neurons fire rhythmically to drive astrocyte somatic Ca²⁺ transients through the octopamine-tyramine receptor (Oct-TyrR)

a, Correlation analyses of neuronal activity and somatic Ca²⁺ transients in astrocytes. Averaged traces of 16 individual astrocytes (GCaMP6s) and 4 pairs of Tdc2⁺ neurites (R-GECO1) from the same sample. Vertical orange bars highlight concomitant activity in astrocytes and neurons (* marked events are shown in inset). Amplitude and duration of individual Ca²⁺ transients in astrocytes correlate highly with activity of Tdc2⁺ neurons. b, Frequency of Ca²⁺ transients in astrocytes (n=10,160 cells total). c,d, Relative changes in numbers of Ca²⁺ transients after acute blockade of octopamine/tyramine signaling by halorhodopsin (c) or terazosin (d) (n=6, 96 cells total). Black dots, when either 561nm light or terazosin was delivered. e, Representative traces of astrocytic Ca²⁺ transients in mutants defective in tyramine/octopamine signaling. f,g, Frequency of Ca²⁺ transients in tdc2^RO54, tβh^nM18 (f, n=6, 96 cells total) and Oct-TyrR^hono mutants (g, n listed for each genotype, 16n cells total). h, Chemotaxis assay (n=12). i, Gentle touch assay (n=30). *p<0.05, **p<0.01, n.s., not significant. Error bar, s.e.m. Paired t-test (c,d), one-way ANOVA followed by Tukey's post hoc test (b,f,g,h,i).

Figure 3. Astrocytes mediate tyramine-induced inhibition of dopaminergic neurons through Oct-TyrR

a, Enhanced activity of dopaminergic neurons in wtrw^ex mutant, Oct-TyrR^hono /+ heterozygotes (n=10, 80 neurites). s, subesophageal segments. t, thoracic segments. b, Tyramine and octopamine (2.5mM) inhibit the activity of dopaminergic neurons (n=6, 48 neurites). c, Astrocyte-specific RNAi for Oct-TyrR or wtrw attenuates inhibition of tyramine on dopaminergic neurons (n=6, 48 neurites). Black dots, when tyramine was perfused. *p<0.05, **p<0.01, n.s., not significant. Error bar, s.e.m. Paired t-test (b, same treatment, c, same genotype), one-way ANOVA followed by Tukey's post hoc test (a,b,c).

Figure 4. Tyramine mediated inhibition of dopaminergic neurons depends on adenosine receptor and glial endocytic function

a, AdoR is required for tyramine mediated inhibition of dopaminergic neurons (n=6, 48 neurites). b, Blockade of endocytic function by dominant negative shibire (shi^DN) attenuates inhibition of tyramine on dopaminergic neurons (n=6, 48 neurites). Black dots, when tyramine was perfused. c, Ca²⁺ influx to astrocytes through TrpA1 is sufficient to inhibit dopaminergic neurons (n=6, 48 neurites). Black dots, when AITC was perfused. d, Model. Olfactory and likely mechanosensory information flow towards Tdc2⁺ neurons that release Oct and Tyr to activate the Oct-TyrR on astrocytes and in turn astrocyte Ca²⁺ entry through the TRP channel Wtrw. Increase in astrocyte Ca²⁺ is sufficient to silence DA neurons through a mechanism requiring the adenosine receptor AdoR, potentially through astrocyte ATP release and its breakdown to adenosine. Inhibition of DA neuron activity by astrocytes is essential for normal chemotaxis and startle-induced reversal behaviors. *p<0.05, **p<0.01, n.s., not significant. Error bar, s.e.m. Paired t test (a,b,c, same genotype), Wilcoxon and Mann-Whitney tests followed by Bonferroni-Holm post hoc test (a,b), one-way ANOVA followed by Tukey's post hoc test (c).