Three- to four-week-old Wistar rats were killed by CO
2 asphyxiation and cervical dislocation in accordance with protocols approved by the University of Auckland Animal Ethics Committee and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Lenses were extracted from the eye and placed into artificial aqueous humor (AAH, in mM: NaCl 149, KCl 4.7, CaCl
2 2.5, glucose 5, and HEPES 5 [pH 7.4] with the osmolarity adjusted to 300 mOsM · kg
−1 with mannitol). With the aid of a dissecting microscope, sharpened forceps were used to gently remove the capsule and the fiber cells attached to it. The capsule with adherent cells was transferred to a tube (Eppendorf, Fullerton, CA) and incubated for 30 minutes in 1 mL of dissociation buffer (Na gluconate 170 mM, KCl 4.7 mM, HEPES 5 mM, glucose 5 mM, 0.125% wt/vol Sigma type 1A collagenase, at 37°C). Cells were gently vortexed before being centrifuged at 1000 rpm for 2 minutes. Pelleted cells were resuspended in 200 μL of AAH, which contained 1 mM GdCl
3. The generic nonselective cation channel inhibitor Gd
3+ was included to prevent the vesiculation of fiber cells that occurs on their isolation.
18 21 The cells were plated onto a poly-
l-lysine coated glass coverslip that formed the bottom of a recording chamber, which in turn was mounted on the stage of an inverted microscope (Eclipse; Nikon, Melville, NY). Once adhered to the coverslip (∼5 minutes) the cells were overlaid with AAH+Gd
3+ and continuously perfused using a peristaltic pump (Minipulse 3; Gilson, Middleton, WI) at a rate of 0.4 to 0.6 mL · min
−1. Hyposmotic AAH was prepared by reducing the NaCl concentration to yield an osmolarity of 250 mOsM · kg
−1. The KCC inhibitor DIOA was purchased from Sigma-Aldrich (St. Louis, MO) and made up to a concentration of 100 μM in either isosmotic or hyposmotic AAH that contained 1 mM Gd
3+ and was introduced to the bath via the perfusion system. In the presence of Gd
3+, DIOA precipitated to give a free concentration of around 6 μM as calculated by spectrophotometric analysis of precipitate formation. Cl
−-free AAH solutions were prepared by equimolar substitution of NaCl with Na gluconate, NaI, or NaNO
3. All solutions were introduced to the bath via the perfusion system. All experiments were conducted at room temperature (∼20°C).