All procedures were approved the University at Buffalo’s Institutional Animal Care and Use Committee.
Loading was done in CBA/CaJ mice (Jackson Laboratories, Bar Harbor, ME) aged postnatal day 16 (P16) to P21. Mice were anesthetized with a dose of 200 mg/kg ketamine: 10 mg/kg xylazine. When the mice were areflexic to paw pinch, they were laid on a warming pad maintained at 37°C (Gaymar Industries INC., Orchard Park, NY) to maintain their body temperature. Puralube Vet Ointment (Pharmaderm, Melville, NY) was applied to protect the eyes. Hair was removed from a small patch of skin posterior to the ear with Nair (), and the skin was sterilized with 70% ethanol and betadine (Purdue products, Stamford, CT). A small amount of 0.5% marcaine (Hospira Inc, Lake Forest, IL) was applied to the skin.
Demonstration of the surgical approach in a euthanized animal. The animals lies on its right side, facing to the right, with its ventral side to the top of the field of view.
Next, we made a small cut on the skin posterior to the pinna with scissors, and then detached muscle tissue from the auditory canal by pulling with forceps (). An incision was made in the auditory canal, and part of the free cartilage was removed (). The tympanic membrane was disrupted and the malleus was removed (). This hole was widened by removing part of the bulla on the posterior side to expose the round window (). The round window was ruptured with a fine insect pin, typically leading to release of clear perilymph. A small hole was drilled just above the line that divides the two turns of the cochlea (, asterisk) using a fine insect pin. At the ages used here, the wall of the upper turn is particularly soft, but gets somewhat harder after P20.
For dye injection, micropipettes were pulled from borosilicate capillary tubes (Sutter Instruments, Novato, CA) using a P-97 micropipette puller (Sutter Instruments, Novato, CA) with a diameter of about 20 μm. The micropipette was attached to the end of a 1 mm syringe. The syringe was attached to a micromanipulator (Narashige, Japan). The tip of the micropipette was loaded with 1 μl of 20% dextran-conjugated fluorescent dye. This consisted of 20% fluorescein () or Texas red () conjugated to 3000-MW-dextran, or 10% of the Texas red-dextran plus 10% calcium green-1 or Oregon green 1 conjugated to 3000 or 10,000 MW dextran (Invitrogen) (). The pipette was lowered into the hole made in the wall of the cochlea and the dye was injected into the cochlea by applying light pressure on the plunger (). After the dye was loaded, the wound was closed using Vetbond (3M, St Paul, MN) and the mouse was injected with 5 mg/kg rimadyl. Mice typically woke 1–2 hr after surgery. After a survival time of 12 hr, mice were sacrificed for brain slicing or anatomy.
GABABR activation reduces presynaptic calcium influx.
Electrophysiology and calcium imaging
Slices in the cochlear nucleus were prepared as described previously (Chanda and Xu-Friedman, 2010b
). Briefly, sagittal sections (160 μm thickness) of the cochlear nucleus were prepared in ice-cold solution containing the following (in mM): 76 NaCl, 75 sucrose, 25 NaHCO3
, 25 glucose, 2.5 KCl, 1.25 NaH2
, 7 MgCl2
, 0.5 CaCl2
. Slices were used for patch-clamping or calcium-imaging using standard recording solution containing the following (in mM): 125 NaCl, 26 NaHCO3
, 20 glucose, 2.5 KCl, 1.25 NaH2
, 1 MgCl2
, 1.5 CaCl2
, 4 Na L-lactate, 2 Na-pyruvate, 0.4 Na L-ascorbate, bubbled with 95% O2
and 5% CO2
. All recordings were made at ~34°C in the presence of 10 μM strychnine. The pharmacological agents used were baclofen (100 μM, Sigma, St. Louis, MO) and CGP55845 (2 μM, Tocris Bioscience, Ellisville, MO).
For electrophysiology controls, BCs were patched under an Olympus BX51WI microscope with a Multiclamp 700B (Molecular Devices) controlled by ITC-18 (Instrutech) interface driven by custom-written software (mafPC) running in Igor (Wavemetrics). Pipettes were 1–2 MΩ, filled with the following (in mM): 35 CsF, 100 CsC1, 10 EGTA, 10 HEPES. The holding potential for voltage clamp was −70 mV with access resistance 3–7 MΩ, compensated to 70%. AN fibers were stimulated using a glass micropipette placed 30–50 μm away from the cell being recorded with currents of 6–14 μA through a stimulus isolator (WPI A360).
For calcium-imaging, labeled fibers were imaged using a 60X water-immersion objective under a 150 W xenon lightsource (Optiquip Model 770), with excitation/dichroic/emission filters of 560DF55/595/645DF75 (for Texas red) or 480DF40/505/535DF50 (for calcium green and Oregon green). Fluorescence was detected using a Sensicam QE (Cooke Corp.) controlled by SIDX drivers (Bruxton Corp., Seattle, WA) running in Igor. Results were filtered with a 7-pixel Gaussian.
After the diffusion step, mice were re-anesthetized, and perfused transcardially with 0.9% NaCl, followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were post-fixed for 2 hour before cryoprotecting overnight in 20% sucrose. Coronal or sagittal sections were made at 40 μm on a sliding microtome (American Optical, Buffalo, NY).
Sections containing the AVCN were collected and washed in 3 5-minute cycles in 0.2 M phosphate buffer saline (PBS), pH~7.6. The sections were then blocked in 0.2 M PBS with 0.1% Triton X-100 (Sigma) and 5% goat serum for two hours. The sections were incubated overnight at 4°C in primary antibody solution containing: anti-CB1 (gift from Mackie lab, Indiana University, Bloomington, IN) at 1:500 concentration, 5% goat serum, and 0.2 M PBS with Triton X-100. The sections were then washed 3 times in 0.2 M PBS solution, and incubated for 2 hours with either Texas-red-conjugated goat anti-rabbit or FITC-conjugated goat anti-rabbit secondary antibody solution (Jackson Immuno Research Laboratories, West Grove, PA) at 1:200 dilution in 0.2 M PBS, 0.1% Triton X-100 and 5% goat serum. The sections were stained with DAPI for cell body labeling, washed in PBS and then mounted on glass slides using Fluoromount G (Southern Biotech). The image in was acquired using a Zeiss AxioImager Fluorescence Microscope.
Average data are presented as mean ± standard error (SE). Statistical significance was determined using the paired, two-tailed, student’s t-test.