Hydrazide

Pressurized arterioles were fixed (2% paraformaldehyde) and stained with: Alexa 633 Hydrazide (0.2 μM, Ex 633/Em 700nm; non-selective matrix staining dye), anti-elastin antibody (1° antibody dilution 1:100; 2° AB, 491/515nm) and a nuclear stain Yo-Pro-1 iodide (491/515nm) or 4′6-diamino-2-Phenylindol (DAPI, 500ng/ml, Ex: 355, Em: 400–450nm) ^{17 (link)}. A subset of vessels were stained for type I collagen using an anti-rat polyclonal antibody (Millipore; 1:1000, 1μg/ml) and a goat anti-rabbit Alexafluor 488 secondary antibody (20μg/ml).
3D confocal microscopy was used to visualize ECM within the arteriole wall. Imaging was performed using a Leica TCS-SP5 microscope in conjunction with a Leica 63X water immersion objective lens (NA 1.2) and Leica LAS AF image acquisition software. 512×512 pixel images were acquired at a resolution of 0.48 μm/pixel at 400 Hz. Line averaging (3 scans) was performed to reduce the low frequency noise. Z dimension step size was 0.05 – 0.3 μm for all z-stacks. All acquisitions represent 8-bit TIFF grey scale images. Post image acquisition analyses were performed using Image J (NIH, Bethesda, MD), Imaris (Bitplane Scientific Software, South Windsor, CT) and Image Pro (Media Cybernetics, Bethesda, MD).
See Supplement for detailed Materials and Methods including additional information relating to image acquisition and processing.

Experiments were conducted at room temperature (22–25°C; unless otherwise noted) using retinal slices (210 µm thick) prepared from homozygous VGLUT3 Cre/YFP mice (P17–21), as previously described (Singer & Diamond, 2003 (link); Chavez et al., 2006 (link)). For experiments involving light stimulation, tissue preparations were conducted under dim red illumination and stored in light-tight containers (to minimize rundown of the cone-driven light response); for all other experiments, tissue preparations were conducted and stored under ambient room lighting (light adapted). Mouse retinas were isolated in artificial cerebrospinal fluid (ACSF) containing (in millimolar): 119 NaCl, 26 NaHCO₃, 1.25 Na₂HPO₄, 2.5 KCl, 2.5 CaCl₂, 1.5 MgSO₄, 10 glucose, 2 Na-pyruvate, 4 Na-lactate (continually bubbled with 95% O₂/5% CO₂). For experiments probing the excitatory synaptic mechanisms (i.e., Figs. 4 and 5), unless otherwise noted, ACSF was supplemented with the group III metabotropic glutamate receptor (mGluR) agonist L-AP4 (10 µM) to mimic dark conditions and strychnine (1 µM), SR95531 (10 µM), and methyl-(1,2,3,6-tetrahydropyridin-4-yl)phosphinic acid (TPMPA, 50 µM) to block glycine-, GABA_A-, and GABA_CRs, respectively. For all nonsynaptic experiments (i.e., assessment of active membrane properties), ACSF was supplemented with L-AP4 (10 µM) and the 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid receptor (AMPAR) antagonist, 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f] quinoxaline −7 sulfonamide (NBQX) (10 µM). Drugs were purchased from Sigma or Tocris (St. Louis, MO) with the exception of tetrodotoxin (TTX) (Alamone Labs, Jerusalem, Israel). Fluorescent dyes were purchased from Molecular Probes (Eugene, OR). VGLUT3+ amacrine cells were visually identified for physiological recordings in retinal slices using either a modified 2PLSM (λ = 930 nm; Zeiss) or standard epifluorescence. When using two-photon laser-scanning microscopy (2PLSM) emissions from the YFP and Alexa 594 were spectrally separated into two distinct collection channels, using a combination of a 565 nm dichroic mirror and 500–550 nm and 570–640 nm band-pass filters (Chroma, Bellows Falls, VT). This allowed for near-independent control of the signals collected from the patch pipette and the cells of interest making high quality recordings relatively easy to achieve. Alternatively, short exposures (typically <30 s) of full-field epifluorescence were used to quickly identify YFP-labeled somas that could then be targeted for electrophysiological recording using infrared differential interference contrast (IR-DIC) video microscopy. Unless otherwise noted, whole-cell voltage-clamp recordings were made from VGLUT3+ amacrine cells using pipettes (~5–6 MΩ) containing (in millimolar): 100 Cs methanesulfonate, 20 tetraethylammonium-Cl, 10 HEPES, 10 EGTA, 10 Na phosphocreatine, 4 MgATP, 0.4 Na-GTP, and 0.04 Alexa 594 hydrazide (pH 7.4). Potassium-based internal for current-clamp recordings contained (in millimolar): 100 K methanesulfonate, 20 KCl, 10 HEPES, 2 EGTA, 10 Na phosphocreatine, 4 MgATP, 0.4 Na-GTP, and 0.04 Alexa 594 hydrazide (pH 7.4). The access resistance for all recordings presented in this study was ≤30 MΩ and remained uncompensated. Input resistance for VGLUT3+ amacrine cells was 1075 ± 477 MΩ (n = 10) when using the potassium-based internal and after correcting for access resistance. Recordings were made using an Axopatch 1D or Axoclamp 700B amplifier (Axon Instruments, Foster City, CA) controlled through an A/D board (Instrutech, Port Washingon, NY) by custom software written in Igor Pro (Wavemetrics, Lake Oswego, OR). All responses were collected at 20 s intervals, low-pass filtered at 5 kHz, and digitized at 10–50 kHz. Voltage steps were leak subtracted using a P/4 subtraction protocol. Isolated excitatory synaptic responses (Figs. 4 and 5) were elicited by electrical stimulation of bipolar cells in the outer plexiform layer (OPL) (~0.5–2 µA for 100–300 µs; FHC, Bowdoin, ME).
For light stimulation, full-field light stimulation was provided by a 470-nm LED (Thor Labs, Newton, NJ) directed through the epifluorescence port of the microscope, band-pass filtered between 450–490 nm, and reflected into the objective by a 510-nm dichroic mirror (Zeiss) to provide full-field illumination over the objective (water immersion 1.0 NA 40X; Zeiss) aperture (0.45 mm diameter). Photon flux at the slice was measured to be approximately 9.6 × 10¹⁸ photons/cm²/s using a DR-2000 radiometer (Gamma Scientific, San Diego, CA). To calculate changes in synaptic conductance evoked by light stimulation, we adapted methods similar to previous reports (Borg-Graham, 2001 ; Taylor & Vaney, 2002 (link); Oesch & Taylor, 2010 (link)). We illuminated the slice for 1 s while holding the cell at a series of command voltages between −90 and 10 mV by increments of 20 mV and recorded current traces as described above, with the exception that these experiments were performed at 35–37°C and 10 mM QX-314 was added to the internal solution to block Na_v channels intracellularly. Offline, we subtracted the leak current after the voltage step prior to the application of light to isolate the light-evoked current and then measured the light-evoked I–V relationship every 10 ms spanning the duration of the light stimulus. We fit each I–V with a line between −90 and 10 mV to extract the slope (g_T) and x-intercept (V_r) for each I–V measurement. If we assume that light-evoked currents (I_e) arise from excitatory and inhibitory synaptic inputs and obey Ohm’s law, so that I_e=g_e(t)(V – V_e) and I_i=g_i(t)(V – V_i), where the inhibitory, g_i(t) and excitatory, g_e(t) conductances are functions of time. The total light-evoked synaptic current is I_T = g_T(t)(V – V_t(t)), where g_T = g_e + g_i. The observed synaptic reversal potential V_r(t) is a weighted sum of V_e and V_i such that, V_r(t) = (g_e(t)/g_T(t))V_e + (g_i(t)/g_T(t))V_i. Then, the excitatory and inhibitory synaptic conductances can be calculated from g_T(t) and V_r(t) according to the equations: g_e(t) = g_T(t){V_r(t) – V_i)/(V_e – V_i) and g_i(t) = g_T(t)(V_r(t) – V_e)/(V_i – V_e). Based on the ionic concentrations used in our internal and external recording solutions, we used a value of 0 mV for V_e and −48 mV for V_i.
Electrophysiology data were analyzed using Igor Pro and Excel (Microsoft). Paired two-tailed t-tests were used to compare data sets and significance was determined as P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***). Unless otherwise indicated, data are presented as mean ± S.D. and illustrated traces are averages of 5–10 responses.