D'Ascenzo et al. 10.1073/pnas.0609408104. |
Fig. 6. Astrocytes in the NAc are dye-coupled. (A) Confocal image showing Alexa 568 (100 mM) spreading from the recorded astrocyte to neighbor cells. (Scale bar: 20 mm.) (B and C) Patch-clamp recording from the dye-filled cell in A shows the passive membrane properties in response to current injection (B) and a linear I-V relationship (C) typical of NAc astrocytes.
Fig. 7. Ca2+-dependent glutamate release from astrocytes generate NMDA receptor-dependent SICs in MSNs. (A Upper) Confocal images of fluo-4 fluorescence in a NAc astrocyte before (first image) and after (images two, three, and four) a UV flash targeted to the cell soma. Acquisition time, 3 s. (A Lower) Simultaneous patch-clamp recording from a MSN 30 mm from the stimulated astrocyte. Note the induction of SIC after the UV flash (arrow). Calibration, 30 mm. (B) Average frequency of SICs in 1 min of a control period before and in an equivalent time period after the photolytic flash (n = 5 cells). (C) Bar graph showing the average rise and decay time of chemically-evoked (open bars, n = 259 events) and photolysis-evoked SICs (filled bars, n = 11 events). (D) Representative SICs (Left) and average SIC frequency (Right) under control conditions, in the presence of D-AP5 (50-100 mM) and after D-AP5 washout. (E) Mean rise, decay time, and amplitude of SICs are not significantly different in the presence (filled bars, n = 259 events) and absence (25 mM; open bars, n = 19 events) of CNQX.
Table 1. Summary of the electrophysiological properties of MSNs and astrocytes in the NAc
| Resting potential, mV | R in, MΩ | AP threshold, mV | AP amplitude, mV |
MSNs | -85.2 ± 1.1 (n = 12) | 226.8 ± 11.9 (n =1 2) | -53.0 ± 1.0 (n = 12) | 75.7 ± 2.3 (n = 12) |
Astrocytes | -89.7 ± 1.4 (n = 15) | 17.8 ± 3.0 (n = 15) | None (n = 15) |
Table 2. Comparison of kinetics of astrocytic evoked SICs and EPSCs triggered by stimulation of glutamatergic afferents
| Amplitude, pA | Rise time, ms | Decay time, ms |
SICs | -120.5 ± 9.3; n = 259 | 81.4 ± 5.8; n = 259 | 451.0 ± 42.2; n = 259 |
EPSCs |
| 1.9 ± 0.2; n = 34 | τ 1 = 8.3 ± 0.4; n = 34τ 2 = 156.1 ± 24.1; n = 34 |
SI Materials and Methods
Electrophysiology.
Slices were visualized with a 40X water immersion objective on an Olympus BX51WI upright microscope equipped with DIC and fluorescence optics. Access resistance was monitored throughout the entire duration of the recording and was typically <15 MΩ. Field recordings were performed with normal ACSF in the pipette. Electrophysiological signals were amplified and filtered at 1 kHz by an Axopatch 1D (Axon Instruments, Union City, CA) or 2400 patch-clamp amplifiers (A-M Systems, Sequim, WA), digitized at 5 kHz with a Digidata 1320 (Axon Instruments), and stored in the computer with Clampex 9.2 (Axon Instruments). Recordings were performed at 30-32°C. Neurons were voltage-clamped at -85 mV and astrocytes at -95 mV. Data were analyzed with Clampfit (Axon Instruments), Excel (Microsoft, Redmond, WA), and SigmaPlot 8.0 (SPSS, Chicago, IL). The interevent time interval between SICs from paired recordings was calculated as the time interval between the onset of the SIC in cell 1 and the onset of the SIC in cell 2. Student's t test was performed to determine statistical significance.Confocal Ca2+ Imaging
. For Ca2+ imaging experiments, slices were incubated under mild stirring for 1 h with 10 mM calcium indicator Fluo-4 AM or Rhod-2 AM (Molecular Probes, Eugene, OR) and 0.15% pluronic F-127. GFP and Rhod-2 fluorescence was excited at 488 and 543 nm, respectively. Fluorescence intensity was measured from individual astrocytes as the average intensity of fluorescence in a region of interest corresponding to the cell soma. The fluorescent signal at a given time point was expressed as DF/F = (F1 - F0)/F0, where F0 and F1 are the value of the fluorescence in astrocytes at rest and at the given time point, respectively.Western Blotting
. The slices of NAc were homogenized by using 100 ml of buffer A containing 20 mM Tris·HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.2% SDS, 50 mM NaF, 1 mg/ml leupeptin, 1 mg/ml antipain, and 0.5 mM phenylmethylsulfonyl fluoride, then the samples were fractionated by SDS/PAGE and analyzed by immunoblotting.Immunocytochemistry.
Animals were deeply anesthetized and intracardially perfused with saline solution followed by 4% of paraformaldehyde. Brains were removed, postfixed overnight, and cryoprotected in 30% sucrose. Free-floating sections were cut at 40 mm in a freezing microtome and kept at -20°C in cryoprotective solution [30% sucrose/30% ethylenglycol/1% poly(vinylpyrrolidone) in PBS] until processing.Sections were washed in PBS and incubated for 2 h in blocking solution (2% normal horse serum/0.5% BSA/0.3% Triton X-100 in PBS) followed by incubation in blocking solution containing the primary antibody (rabbit anti-mGluR5; Chemicon) for 48 h at 4°C. After rinsing in PBS, sections were incubated in blocking solution containing biotinylated anti rabbit antibody for 2 h at room temperature, then rinsed in PBS and incubated 15 min in PBS containing Texas Red Avidin DCS (Vector Laboratories). After washing, sections were mounted by using VECTASHIELD mounting medium (Vector Laboratories).
Sections were visualized on an Olympus IX81 microscope combined with an Olympus FV1000 confocal microscope. Excitation at 488 nm for GFP and 543 nm for Texas red was provided by Ar and HeNe ion lasers, respectively. Fluorescence emission was collected at 500-530 nm for the GFP (channel 1) and 555-655 nm for Texas red (channel 2). Images were acquired with Olympus FluoView 1.3 and analyzed with MetaMorph and Volocity 3.6 software. Background fluorescence for channel 1 was measured in slices from GFP-GFAP-negative mice and for channel 2 from slices incubated with only the secondary antibody. A binarized image of channel 1 (GFP) was then used as a mask to identify mGluR5 immunoreactivity that colocalized with GFP.
Two-Photon Microscopy.
Two-photon imaging was performed by using an Ultima scanhead (Prairie Technologies, Middleton, WI) attached to an Olympus BX51WI microscope equipped with a ´60 water-immersion objective. Excitation for GFP and Rhod-2 was provided at 820 nm, and emission was detected by external photomultiplier tubes (525/70; DLCP 575; 607/45 nm).Flash Photolysis.
Photolysis experiments were performed as described by Fellin et al. [Fellin T, Pascual O, Gobbo S, Pozzan T, Haydon PG, Carmignoto G (2004) Neuron 43:729-743]. Briefly, NAc slices from young mice (9-14 days old) were loaded for 1 h at room temperature in ACSF containing Fluo-4 AM (12.5 mg/ml), NP-EGTA AM (25 mg/ml), DMSO (0.1%), and pluronic (0.05%) saturated with 95% O2/5% CO2. This resulted in the selective loading of astrocytes with the Ca2+ indicator fluo-4 and the calcium cage NP-EGTA. The fluorescent dye Alexa 568 (100 mM) (Molecular Probes) was added to the intrapipette solution used for electrophysiological recordings to visualize the cell body and the dendrites of the recorded neuron. Photorelease of Ca2+ was performed by using a 3-mm-diameter UV pulse (351 and 364 nm) generated by an argon ion laser (Coherent Enterprise II; duration 100 ms, power 200-250 mW) connected by an optical fiber to an Uncager system (Prairie Technologies).Drugs.
D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), ifenprodil hemitartrate, (-)-quinpirole hydrochloride, (RS)-3,5-DHPG, 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline disodium salt (CNQX), baclofen, SKF 38393 hydrobromide, and 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) were purchased from Tocris Cookson. Fluo-4-AM and Rhod-2-AM were from Molecular Probes. [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydro-quinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) was supplied by Novartis.