Fig. 1.

NAc astrocytes are electrically inexcitable, are electrotonically coupled with one another, express mGluR5 receptors, and respond to neurotransmitters with Ca²⁺ oscillations. (A) Two-photon laser-scanning image of GFP-expressing cells from a NAc slice obtained from pGFAP-GFP transgenic mice (z-projection of 15 planes). (Scale bar: Upper, 40 μm; Lower, 10 μm.) An astrocytic endfoot making contact with a capillary (∗). (B) Typical response of a NAc pGFAP-GFP⁺ cell to current injections from −0.2 to 1.1 nA (bottom traces, 0.1-nA increments). (C) Average whole-cell I–V relationship from GFP-expressing cells (n = 15 cells). (Inset) Inward and outward currents recorded in voltage-clamp configurations (−135 to +25 mV, 10-mV increments). (Scale bars: 2 nA, 50 ms.) (D) Paired recordings from two GFP-positive cells showing bidirectional coupling. (E–G) mGluR5 is expressed in NAc astrocytes. Top-view reconstruction obtained from confocal z-series of GFP-expressing astrocytes (E, green). mGluR5 immunoreactivity (F, red) associated with GFP-positive astrocytic processes reveals localization of this receptor mainly in astrocytic processes rather than in astrocytic cell bodies (G). [Note that we used an image mask based on the GFP fluorescence of the astrocyte to display pixels displaying mGluR5 immunoreactivity associated with astrocytes (see SI Materials and Methods).] Insets in G show regions 1 and 2 at higher magnification. (Scale bars: E–G, 20 μm; G Inset, 10 μm.) (F Inset) Western blot analysis from isolated NAc slices confirm the presence of the mGluR5 in this brain region. (H) NAc slices from pGFAP-GFP mice loaded with Rhod-2 (red) showing selective labeling of GFP expressing astrocytes (green). (Scale bar: 20 μm.) (I) Current-clamp recordings from a dye-loaded cell showing astrocytic membrane properties and no action potential firing. (J) Average whole-cell I–V relationship from dye-loaded cells (n = 3 cells) and representative currents after voltage steps from −115 mV to +25 mV (Inset). (K) Astrocyte responses (time course of the ΔF/F ratio) to DHPG (20 μM; Upper) and ATP (100 μM; Lower). (L) Average percentage of astrocytes displaying Ca²⁺ oscillations under the different experimental conditions: ACSF, n = 23 slices; DHPG (20 μM), n = 4 slices; DHPG (20 μM) + MPEP (50 μM), n = 4 slices; ATP (50/100 μM), n = 5 slices; baclofen (40 μM), n = 4 slices; low Ca²⁺, n = 5 slices; SKF 38393 (2.5–5 μM), n = 3 slices; quinpirole (20 μM), n = 3 slices; and cocaine (1–10 μM), n = 4 slices.