Synaptic and extrasynaptic factors governing glutamatergic retinal waves.

Animals

C57Bl/6 mice obtained from Harlan were used for all WT recordings. VGLUT1 knockout mice ( Fremeau et al., 2004 ) and VLGUT3 knockout mice ( Seal et al., 2008 ) were generated as reported previously. Heterozygous littermates were used as controls for experiments using VGLUT1- and VGLUT3-null mice. All animal procedures were approved by the University of California, Berkeley; University of California, San Diego; or University of California, San Francisco Institutional Animal Care and Use Committees and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals , the Public Health Service Policy, and the Society for Neuroscience Policy on the Use of Animals in Neuroscience Research. Whole-Mount Retinal Preparation P10 – P24 mice were anesthetized with halothane or isoflurane and decapitated. Retinas were isolated in artificial cerebrospinal fluid (ACSF) (in mM: 119.0 NaCl, 26.2 NaHCO 3 , 11 glucose, 2.5 KCl, 1.0 K 2 HPO 4 , 2.5 CaCl 2 , 1.3 MgCl 2 ) and mounted RGC side up on filter paper. Retinas were incubated at room temperature in bubbled (95% O2/5% CO2) ACSF until transfer to the recording chamber, where they were constantly superfused with bubbled ACSF (32–34°C unless otherwise specified).

Electrophysiology and Pharmacology

Whole-cell current and voltage clamp recordings were made from whole-mount retinas. Retinas were visualized with differential interference contrast optics on a Zeiss Axioskop 2 FS Plus microscope with an Achroplan 40x water immersion objective. The inner limiting membrane of the retina was removed with a glass recording pipette and RGCs were targeted under control of a micromanipulator (MP-225; Sutter Instruments). RGCs were identified by their large somas and the presence of a voltage-gated sodium conductance. Recording pipettes (Garner Glass or Sutter Instruments) were pulled (PP-830; Narishige) with a tip resistance of 3–6 MΩ and filled with either a cesium gluconate (for all voltage-clamp recordings; in mM: 100 CsOH, 100 gluconic acid, 1.7 CsCl, 40 HEPES, 10 EGTA, 5 MgCl 2 , 1 QX-314, 2 Na 2 ATP, and 0.3 Na-GTP; pH adjusted to 7.25 with CsOH; E Cl = −60 mV) or potassium gluconate internal solution (for current-clamp recordings; in mM: 98.3 K-gluconate, 40 HEPES, 1.7 KCl, 0.6 EGTA, 5 MgCl 2 , 2 Na 2 ATP, and 0.3 Na-GTP; pH adjusted to 7.25 with KOH). Data were acquired using pCLAMP 9 recording software and a Multiclamp 700A amplifier (Molecular Devices), sampled at 5 kHz and low-pass filtered at 1 kHz. 6,7-Dinitroquinoxaline-2,3-dione disodium salt (DNQX), D-(−)-2-Amino-5-phosphonopentanoic acid (AP5), 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo [f]quinoxaline-7-sulfonamide disodium salt (NBQX), SR-95531 hydrobromide (gabazine), strychnine, and Dihydro-β-erythroidine hydrobromide (DHβE) were added to ACSF as stock solutions prepared at ≥1000x concentration in water. DL-threo-b-Benzyloxyaspartic acid (TBOA) stock solution was prepared at 1000x in dimethyl sulfoxide. All antagonists were acquired from Tocris; all other chemicals were acquired from Sigma-Aldrich. Data were analyzed in pCLAMP, IGOR Pro (Wavemetrics Inc.) or MATLAB (Mathworks). To calculate inter-cEPSC intervals in whole-cell voltage clamp recordings, cEPSCs were identified by eye. For Figure 2 , a cluster was defined as a series of cEPSCs separated by not more than 10 s. Outside-out patches were excised from RGC somas > 500 μm away from the simultaneously recorded RGC. Cesium gluconate was used for outside-out patch recordings. To confirm that patches contained glutamate receptors, L-glutamate was pressure-applied (5 psi) to patches at a concentration of 1mM in a solution containing (in mM): 150 NaCl, 2.5 KCl, 10 Hepes, pH 7.4. Patches were inserted to a depth of 10 μm in the inner plexiform layer, within 10 μm of the recorded RGC’s soma. All patch traces included were low-pass filtered (Bessel, 8-pole) at 150 Hz for clarity.

Calcium Imaging

Retinas were bulk loaded with the calcium indicator Oregon Green 488 BAPTA-1 AM (OGB-1 AM; Invitrogen) using the multicell bolous loading technique ( Stosiek et al., 2003 ). OGB-1 AM was prepared at a concentration of 10mM in a solution of 2% pluronic in DMSO, which was then diluted 1/10 in a solution containing (in mM): 150 NaCl, 2.5 KCl, 10 Hepes, pH 7.4 ( Stosiek et al., 2003 ). The OGB-1 AM solution was pressure ejected from a borosilicate glass micropipette (Garner Glass or Sutter Instruments) using a PV-820 Pneumatic PicoPump (World Precision Instruments) at a pressure of 10 – 20 psi. The pipette was positioned just under the inner limiting membrane and dye was injected for 700 ms in 3–5 locations per retina. Epifluorescent calcium imaging was performed on a Zeiss Axioskop 2 FS Plus microscope using a 10x or 40x water immersion objective (Zeiss Achroplan), with illumination provided by a Sutter Lambda LS and controlled by a Uniblitz shutter. Images were acquired at 2–4 Hz in MetaMorph (Universal Imaging Corporation).

Statistics

Paired t-tests were used for all paired comparisons. For multiple comparisons, a repeated measures ANOVA with a Newman-Keuls post hoc test was used. Levene’s test was used to compare variances. Alpha was always 0.05.

Supplementary Material Supplemental Experimental Procedures Supplemental Movie 1 Supplemental Movie 2 Supplemental Figure 1 Supplemental Figure 2 Supplemental Figure 3 Supplemental Figure 4