PMC

Astrocyte response to SST interneurons does not depend on glutamatergic transmission. a Representative traces and b–d quantitative evaluation of GCaMP6f-astrocyte responses to SST interneuron activation (10 or 30 light pulses) in SSCx slice preparations (9 astrocytes, 9 slices, 3 mice) in the presence of NBQX (10 µM), D-AP5 (50 µM), and MPEP (50 µM). Scale bars, 20 s, 50% dF/F₀. Data are means ± SEM

The neuropeptide somatostatin prevents the depression of the astrocyte response to PV interneurons. a–c Average data of GCaMP6f-astrocyte Ca²⁺ response to PV interneuron activation (10 or 30 light pulses) from SSCx slice preparations (8 astrocytes, 8 slices, 3 mice) in the presence of somatostatin (1–2 μM). d Cumulative distributions of Ca²⁺ events show no response depression to successive 30 pulse stimulations of PV interneurons (for proximal processes, p = 0.694; for microdomains, p = 0.957, Kolmogorov–Smirnov test)

Specificity of neuropeptide-releasing interneuron signaling to astrocytes. a Astrocyte response ratio (RR, see Methods) of the second versus the first 10 or 30 light pulse stimulation of PV or SST interneurons in the absence or presence of the neuropeptide SST or of a somatostatin receptors (SSTRs) blocker. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Data are represented as mean ± SEM. b Schematic of the signaling specificity to astrocytes of SST and PV interneurons (left) and of the potential recruitment of the astrocytic network by different neuropeptide-releasing interneurons (right)

GABA_BRs and SST4Rs colocalize at non perisynaptic astrocytic processes (nPAPs). a Upper row, GABA_B2 and SSTR4 immunogold EM single-labeled nPAPs (arrows point to 18 and 12 nm membrane-associated gold particles, respectively). Middle row, GABA_B2-SSTR4 double-labeled PAPs nearby to an axo-dendritic shaft (left) and an axo-spinous (right) symmetric synapse (arrowheads point to edges of active zones). Lower row, double-labeled nPAPs not in the proximity of symmetric synapses (arrowheads). b GABA_B2 and SSTR4 relative density (particles/μm²) in double-labeled PAPs (n = 40) and nPAPs (n = 77) is higher than background (BG; p < 0.0001, Mann–Whitney test), whereas it is similar between PAPs and nPAPs. c Distance distribution of immunogold GABA_B2 and SSTR4 pairs at PAP membranes (40 pairs from 37 PAPs) and nPAPs (100 pairs from 97 nPAPs). d Lateral position of GABA_B2 and SSTR4 pairs with an edge-to-edge distance within 50 nm with respect to the closest AZ margin of a symmetric synapse (53 couples from 53 nPAPs). Axt, axon terminal; Den, dendrite; s, spine. Scale bars, 100 nm

Properties of the astrocyte response to PV and SST interneurons in vivo. a–c Average data of Ca²⁺ signal dynamics at different compartments of GCaMP6f-astrocytes from ChR2-PV- or ChR2-SST-GCaMP6f mice in response to PV interneuron (14 astrocytes, 7 mice) and SST interneuron signaling (20 astrocytes, 8 mice). d, f Cumulative distributions of astrocytic Ca²⁺ events confirming significant response depression to successive PV interneuron stimulations and significant response potentiation to successive SST interneuron stimulations. *p ≤ 0.05, **p ≤ 0.01; ***p ≤ 0.001, Kolmogorov–Smirnov test. Exact p-values for the data reported in this as well as all the other figures are reported in the Supplementary Table . Mean percentage of responsive astrocytes, mean frequency and amplitude of somatic Ca²⁺ events in response to PV interneuron (white bars, 100 astrocytes, 6 ChR2-PV mice) or SST interneuron (black bars, 90 astrocytes, 7 ChR2-SST mice) optogenetic activation in the SSCx in vivo after loading with OGB-1 and the specific astrocytic marker SR101. Data are represented as mean ± SEM

SST interneuron signaling specificity to astrocytes depends on the neuropeptide somastostatin. a Representative traces and b, c quantitative evaluation of GCaMP6f-astrocyte responses to the neuropeptide SST in SSCx slices in the absence (28 astrocytes, 8 slices, 3 mice) or the presence (9 astrocytes, 9 slices, 3 mice) of antagonists of GABA_B (SCH50911, 50 µM), mGlu5 (MPEP, 50 µM) and purinergic (PPADS, 100 µM) receptor antagonists and in the continuous presence of TTX (1 µM). Scale bars, 50 s, 20% dF/F₀. The response of GCaMP6f-astrocytes is not affected by the mix of receptor antagonists. d Representative Ca²⁺ traces of a GCaMP6f-astrocyte before and after subsequent 10 and 30 pulse SST interneuron activations in the presence of 20 μM CYN 154806. Scale bars, 50 s, 20% dF/F₀. e Mean number of active ROIs, event frequency and amplitude at soma, proximal processes and microdomains of GCaMP6f-astrocytes (57 astrocytes, 10 slices, 5 mice). Data are represented as mean ± SEM

Calcium signal dynamics reveal differential astrocyte responses to PV and SST interneuron activation. a Schematic of the in vivo experimental approach (left) and of the optogenetic stimulation of ChR2-PV or ChR2-SST interneurons (right). b Top, images of a representative GCaMP6f-astrocyte in layer 2/3 SSCx from an adult ChR2-PV-GCaMP6f mouse with the ROIs defined by GECIquant software for the Ca²⁺ response to the first 30 light pulse stimulation (blue lines) of PV interneurons at the soma (yellow), proximal processes (red), and microdomains (blue), scale bar, 20 μm (see Supplementary Movie ). Bottom, Ca²⁺ signal dynamics at different astrocytic compartments before and after successive 10 and 30 light pulse PV interneuron activations. Scale bars, 50 s, 20% dF/F₀. c, d Raster plots of Ca²⁺ peaks (c) and mean time course of Ca²⁺ transients (d) from all in vivo monitored GCaMP6f-astrocytes, at rest and following PV interneuron stimulations. Scale bar, 5 s, 20% dF/F₀. e–g Same as in b–d, but for ChR2-SST-GCaMP6f mice and SST interneuron stimulation

GABAergic signaling from individual PV or SST interneurons in SSCx slices is sufficient to recruit neighboring astrocytes. a, b Top, schematics of patch-clamp experiments and representative AP firing induced by intracellular depolarizing current pulse injections into a PV (mean firing rate, 11.8 ± 2.1 Hz) or a SST interneuron (mean firing rate 9.0 ± 1.44 Hz; burst firing rate, 30 ± 4.8 Hz). Scale bars, 100 ms, 20 mV, 200 pA. Bottom: somatic Ca²⁺ signal dynamics from representative astrocytes before and after two sequences of 30 current pulses (red lines) delivered to PV or SST interneurons in absence or presence of SCH50911 (SCH, 50 µM). Astrocytes from an area within 100 μm from the patched interneuron were considered. Scale bars, 50 s, 20% dF/F₀. c Mean percentage of responsive astrocytes and mean Ca²⁺ oscillation frequency in response to individual PV (65 astrocytes, 4 slices, 4 mice) or SST interneuron (62 astrocytes, 4 slices, 4 mice) stimulation. Data are represented as mean ± SEM. d Cumulative distributions of Ca²⁺ event frequency after two subsequent 30 current pulse injections. With respect to the first stimulation, the astrocyte response to the second PV interneuron stimulation was significantly depressed, whereas that to the second SST interneuron stimulation was significantly potentiated (Kolmogorov–Smirnov test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)