Sagittal olfactory bulb slices (280–350 μm thick) were prepared from young transgenic V2r1b mice (Del Punta et al., 2002
) post-natal day 0 to 33 as described previously (Urban and Castro, 2005
). Mice were anesthetized (0.1% ketamine/0.1% xylaxine; ~ 3mg/kg, i.p.) and decapitated. Olfactory bulbs were removed and sectioned on a Leica vibratome (Germany) while submerged in ice-cold oxygenated Ringer’s solution containing the following (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3
, 1.25 NaH2
, 25 glucose, 2 CaCl2
. All procedures were in accordance with the guidelines of Institutional Animal Care and Use Committee of Carnegie Mellon University.
Whole-cell voltage recordings were obtained from the somata of identified AOB mitral cells. Slices were superfused with oxygenated Ringer’s solution containing the following (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 1MgCl2, 25 glucose, 2 CaCl2, warmed to 34–36° C. Whole-cell recordings were established using pipettes (resistances of 2–8 MΩ) filled with a solution containing the following (in mM): 120 potassium gluconate, 2 KCl, 10 HEPES, 10 sodium phosphocreatine, 4 MgATP, and 0.3 Na3GTP, adjusted to pH 7.3 with KOH. Voltage and current clamp recordings were performed using a MultiClamp 700A amplifier (Molecular Devices, Union City, CA). Data were low pass filtered (4 kHz) and digitized at 10 kHz using an ITC-18 (Instrutech, Mineola, NY) controlled by custom software written in Igor Pro (Wavemetrics, Lake Oswego, OR). In some experiments in which membrane current was recorded, mitral cells were held at −90 mV to facilitate the recording of glutamate-mediated EPSCs. Otherwise, the neurons were maintained at resting membrane potential (−50–60mV).
Urine Collection and Purification
Urine was collected from male and females separately that were 3–9 months old housed in metabolic cages and was stored at −20°C until use. Urine was then thawed and centrifuged at 500-g for 5 minutes and the supernatant was filtered through 0.2 μm filter paper. Female and Male urine were mixed in a 1:1 ratio for all experiments. Urine was then purified in 12-mL Sep-Pak Discovery DSC-18 Cartridges (Supelco, Bellefonte, PA). Cartridges were primed with 3 ml of methanol followed by washing with 5 ml of di-water with 0.1% TFA before addition of the urine samples onto the column. Urine samples were then washed with 1 ml of 2% NaCl in TFA. Finally, the peptide was eluted using consecutive additions of 1 ml of 75% Acetonitrile/ 25% Water with 0.1% TFA, (5X) with each fraction collected in conical tubes. In order to determine which fractions contained the peptide of interest, pure urine samples were spiked with derivitized pure peptide to observe, via fluorescence, which fractions contained the peptide.
Peptide Stock Concentration Determination and Derivitization
Stock concentrations of pure peptide (Genscripts) were made by dissolving ~5mg of peptide in H2O. Protein concentration was determined spectrophotometrically using a Thermo Scientific NanoDrop ND-1000 spectrophotometer (Wilmington, DE) assuming a molar extinction coefficient of 1280 cm−1 M−1 (λ = 280 nm) using the Beer-Lambert Law (A = εlc). The peptide was derivitized by adding 10 μl of dansylchloride solution (2.5 mg/ml in acetonitrile) followed by incubation at 37o for 30 minutes.
Lyophilization and Reversed Phase Liquid Chromatography
Fractions containing the peptide of interest were frozen at −80°C and lyophilized using a Labconco Freezone 4.5 freeze dry system (Kansas City, MO), and the resulting solid was reconstituted in 100 μl of water with 0.1% acetic acid. Each sample was then run through High Performance Liquid Chromatography (HPLC) on an Alliance Waters 2695 separation module (Milford, MA) using a C18 Symmetry 300 Column (3.9 mm × 150 mm, 5 μM beads). Gradient elution was carried out at a flowrate of 1.0 ml/min with a mobile phase from 0 to 50% of 0.1% TFA in acetonitrile vs. 0.1% TFA in H2O in 20 minutes. Fluorescence of the eluate was monitored with a Waters 996 Photodiode Array Detector using an emission wavelength of 220 nm. Fractions were collected about a minute and a half before and after the peak. The resulting fractions were lyophilized again and reconstituted in 50 μl of 0.1% acetic acid in H2O for LC-MS.
Liquid Chromatography Electrospray Ionization Mass Spectrometry/Mass Spectrometry
Peptide confirmation and quantification was performed by running the samples on a Michrom BioResources, Magic 2002 HPLC through a Magic MS C18 column (1.0 mm × 150 mm, 5 μM beads) using a gradient elution with a flow rate of 75 μl/min. The gradient went from 0 to 40% mobile phase B in 40 minutes where B equaled 99% acetonitrile with 0.1% acetic acid while mobile phase A equaled 99.9% H2O with 0.1% acetic acid. ESI-MS/MS was performed using a Thermo-Fisher LCQ ESI/APCI ion trap mass spectrometer for the parent scan of m/z 553.7 fragmentation to m/z 251.1, 398.1, 709.3, and 856.4 in a multiple reaction monitoring scan. The isolation width on m/z 553.7 was Δm/z= 3.0 with a collision energy of 31%. Chromatogram peaks were filtered based on the MS/MS fragmentation and were averaged and smoothed using a 7-point Gaussian filter, then integrated using Thermo Fisher Xcalibur Qual Browser software Ver. 1.3 and plotted vs. concentration to form a calibration curve for quantitative analysis. A linear regression of this curve was fitted and extrapolated to determine peptide concentration in pure urine samples. Both spiked urine samples and pure urine samples were prepared, purified, and quantified the exact same way.
VNO and AOB Tissue Collection and Immunohistochemistry
For animals painted with Alexa 594 hydrazide (MW = 758 Da), the dye was made at 1mM in internal buffer solution containing the following (in mM): 150 D-gluconic acid, 10 HEPES, 2 KCl – pH adjusted to 8.05 with KOH. Animals at all ages were transcardially perfused first with 0.1 M phosphate buffer (2 minutes) then with 4% PFA (2 minutes), and their brains were dissected and fixed overnight at 4 C before being transferred to a 0.1M phosphate buffer solution (pH = 7.2) containing 30% sucrose. The heads were also fixed overnight in 4% PFA before being transferred to 0.1M phosphate buffer (pH = 7.2). Each brain was sectioned at 25 μm using a Leica cryostat SM2000R (Germany) and the sections placed in 1 ml of 0.1M PB solution containing 0.05% Na azide and 0.005% Tween 20. The heads were mounted against a 4% agarose block and sectioned at 200 μm using a vibratome and the sections were also transferred to 24 well plates containing 1 ml of PB solution.
For anti-GFP staining, both VNO and brain sections were permeabalized in 2% normal donkey serum and 2% TX-100 and then washed 3X with PB incubating for 5 minutes each wash. The solution was then aspirated and replaced with the primary antibody(300 μl of 1:1000 GFP Mouse antibody, Invitrogen) in PB with 2% normal donkey serum and 0.01% TX-100. After washing 3X again in PB, the solution was aspirated and replaced with the secondary antibody (300 μl of 1:1000 donkey antibody mouse conjugated 488, Invitrogen) with 2% normal donkey serum and 0.01% TX-100 and incubated for 1 HR in the dark and finally washed 3X in PB. The sections were then mounted in gelvatol for confocal imaging and analysis.
For anti-c-Fos staining, sections were permeabilized in PB containing 2% normal donkey serum (NDS, Jackson ImmunoResearch, West Grove, PA) and 0.01% Triton X-100 Sigma) for 1 HR at RT. After three washes in PB, sections were labeled with mouse anti-c-Fos (Calbiochem), 1:1000 in PB with 2% NDS and 0.05% Tween 20 for 1 HR at RT. Following three washes in PB, sections were labeled with donkey anti-mouse AlexaFluor 488 (Invitrogen) 1:600 in PB containing 2% NDS and 0.05% Tween 20 with 1:40K Hoechst 33342 (Invitrogen) for 1 HR at RT in the dark. After a final three washes in PB, sections were mounted on collagen-coated slides with gelvatol for imaging and analysis.
VSN Axonal Coalescence Imaging and Analysis
Confocal image stacks were acquired using a Zeiss LSM 610 microscope using a 40X oil objective. Image stacks were median filtered using ImageJ software with a Gaussian width of 2.0 pixels. For analysis of axonal arborization, maximum intensity z-projections of each stack were imported into custom written software in Igor Pro. Individual glomeruli were analyzed by placing the coordinates of a semi-circle such that the arc of the semi-circle was oriented ventral to the section with the straight-edge cutting off the axons just above the glomerulus. This ensured that each concentric semi-circle expanded throughout the axonal terminations in the glomerulus while excluding the axons superficial to the glomerulus. Sholl analysis was performed such that the final concentric semicircle encompassed the entire glomerulus. Histogram plots of the number of pixels crossing threshold as a function of distance were calculated and the width of the peak of each histogram plot was measured at half of the maximum amplitude.
Mitral Cell Reconstruction and Tuft Analysis
Cells were filled with Neurobiotin (Vector Labs, Burlingame, CA), during whole-cell recordings and slices were placed individually in a well of a 24 well plate (Corning, New York) containing 1 mL of 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.2, and stored at 4 C for up to two weeks. After washing twice with phosphate buffer pH 7.2 (PB), endogenous peroxidase was quenched with 1% H2O2 (Sigma, St. Louis, MO) and 10% absolute MeOH in PB at RT until bubbling stopped (approximately one to three hours). After washing twice with PB at RT, slices were permeabilized for 1 HR at RT with 2% Triton X-100 (Sigma) in PB. During this incubation, Vectastain (Vector Labs) was prepared (using the dropper bottles provided) by adding two drops of Reagent A and two drops of Reagent B to 5 mL of PB containing 1% Triton X-100. The permeabilization buffer was removed at the end of the incubation period and replaced with the Vectastain reagent. Slices were then incubated overnight at 4C in Vectastain. The next day, slices were washed twice for 10 min, twice for 15 min, and once for 1 HR with PB at RT. During the last wash, a 0.5 mg/mL solution of 3,3’-diaminobenzidine tetrahydrochloride (DAB) was prepared by dissolving one DAB tablet (Sigma) in 20 mL of PB. Immediately before use, 6.7 uL of H2O2 was added, and then slices were incubated in the DAB solution for up to 10 min with constant monitoring of color development. At the end of the DAB reaction, slices with washed 5X for 10 min each in PB at RT. They were then mounted on collagen-coated slides in gelvatol using a Parafilm spacer between the slide and coverslip. The sections were then examined under DIC and their dendrites were reconstructed using Neurolucida 8.11(MicroBrightField) and analyzed using Neurolucida Explorer (MicroBrightField). Density calculations were performed in Matlab using XYZ coordinates of traced mitral cells in Neurolucida. Sliding windows of 5, 25, and 50μm were used and a density histogram was calculated for each of the window sizes. The mean of the histogram at two standard deviations above the mean was calculated in order to compare across age groups.
Slices containing AOB were examined under fluorescence (488nm) to identify GFP-positive glomeruli innervated by V2r1b-GFP expressing sensory neurons. Glass electrodes were pulled to a tip size of 3–7μm and filled with 5–10% solutions of Dextran conjugated Alexa 594 (Molecular Probes). The tip of the electrode was placed in the center of a GFP-positive glomerulus. The electroporation protocol consisted of 1200 pulses, (25msec duration, 1–2μA), at 2 Hz were given by an SIU box controlled by TTL pulses from the ITC-18 data acquisition board as described in (Hovis et al., 2010
). This typically resulted in fluorescent dye loading of 6–10 mitral cell bodies and their dendrites per slice. This tissue was fixed in 4% paraformaldahyde for approximately 24 hours and examined post-hoc using confocal imaging (LSM 610 Meta Axioscope 2). Single images, image stacks, and 3D reconstructions of slices were taken and compiled using Zen 2007 software.