In vivo experiments took place at the University of California, San Francisco under a Committee on Animal Research (CAR) approved protocol. In vitro experiments were performed at New York University School of Medicine in conformity with local Institutional Animal Care and Use Committee (IACUC) regulations.
mice were generated in a CD1 genetic background (Ma et al., 1997
). These mice lack detectable AQP4 protein and phenotypically had normal growth, development, survival, and neuromuscular function. Brains from wild type and AQP4-/-
mice showed no gross anatomical differences (Manley et al., 2000
). Male AQP4+/+
mice were used with matched age and body weight (3-4 months, 30-35 g for the in vivo
study and 4-4.5 months, 35-46 g for the in vitro
). Twenty mice were used: seven AQP4 +/+
and seven AQP4 -/-
for in vivo
studies and three AQP4 +/+
and three AQP4 -/-
mice for in vitro
In vivo animal preparation
Mice were anesthetized using 2.5% Avertin (2,2,2-tribromoethanol, 250 mg/kg, i. p., Sigma-Aldrich, St. Louis, MO) and immobilized in a stereotaxic apparatus. An additional 85 mg/kg Avertin was given every hour to maintain anaesthesia. After exposing the skull by midline skin incision, an atraumatic craniectomy was made over the somatosensory cortex (1.5 mm lateral and 1.5 mm caudal to bregma) using a micro drill and the dura was carefully removed. The skin flaps were held open with a cylindrical plastic dam (13 mm diameter and 4 mm height; ) and artificial cerebrospinal fluid (ACSF; see below), at 37°C, superfused over the brain at 2 ml/min. A small plastic container (4 mm diameter and 5 mm height) was glued to the inner wall of the dam to hold agarose gel for control measurements. The mice breathed room air spontaneously and a heating pad maintained body temperature at 37 ± 0.5°C. Measurements were made at 1-3 hours after surgery. The ACSF composition was (in mM): 126 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 10 D-glucose, 1.3 MgCl2, 1.5 CaCl2, gassed with 95% O2/5% CO2 to buffer pH at 7.4. For TMA+ calibration, 0.5 mM TMA-chloride was added.
Figure 1 Diffusion of TMA+ in the somatosensory neocortex of AQP4+/+ and AQP4-/- mice in vivo. A. Photograph of recording arrangement for RTI-TMA diffusion measurements. The mouse head was immobilized in a stereotaxic frame. A micromanipulator positioned the microelectrodes (more ...)
In vitro brain slices
Neocortical slices were prepared as described previously (Hrabětová et al., 2003
). Briefly, animals were deeply anesthetized with sodium pentobarbital (50 mg/kg, i. p.), decapitated and the brain was removed from the skull and cooled with ice-cold ACSF. Coronal sections were cut at 400 μm thickness with a vibrating blade microtome (VT 1000 S; Leica Instrument GmbH, Nußloch, Germany), submerged in ACSF and incubated at room temperature for 1-6 hours before measurements. The composition and gassing of the ACSF were the same as described above except that NaCl was 124 mM and KCl was 5 mM.
For measurements, a brain slice was transferred to a submersion tissue chamber (model RC-27L; Warner Instruments, Hamden, CT) perfused at 2.0 ml/min with ACSF warmed to 33 ± 1°C. The chamber was mounted on a Burleigh Gibraltar stage (EXFO Life Sciences, Ontario, Canada) attached to an Olympus BX61WI microscope (Olympus America, Melville, NY).
The RTI- TMA method
Microelectrodes for iontophoretic delivery and TMA+
-ion-selective microelectrodes (ISMs) were both pulled from double-barrelled theta-glass (Cat. No. 64-0811, Warner Instruments) with a final o.d. of 2-5 μm, and fabricated as described by Nicholson (1993)
. A tetraphenylborate-based ion exchanger (Corning 477317, currently available as IE190 from WPI, Sarasota, FL) was used in the ion-sensing barrel, which was backfilled with 150 mM TMA-chloride. The reference barrel contained 150 mM NaCl. To extract the TMA+
signal, the voltage measured by the reference barrel was subtracted from the voltage measured by the ion-detecting barrel using a dual-channel microelectrode preamplifier (Model IX2-700; Dagan Corp., Minneapolis, MN). Each ISM was calibrated in solutions containing 0.5, 1, 2, 4, 8 mM TMA-chloride in 150 mM NaCl, using the Nikolsky equation. The iontophoresis microelectrode contained 150 mM TMA+
Diffusion measurements in vivo
The shank of the iontophoretic microelectrode was bent and aligned parallel with the ISM and both were glued together using dental cement with an inter-tip distance of 100-150 μm (). Control TMA+
diffusion curves were first recorded in agarose gel (0.3% NuSieve GTG, FMC BioProducts, Rockland, ME, in 150 mM NaCl and 0.5 mM TMA-chloride) to obtain the transport number nt
of the iontophoresis microelectrode and confirm the microelectrode spacing r
, then measurements were made in brain to obtain the effective diffusion coefficient D
, and the constant k
’ that accounted for non-specific loss of TMA+
from the ECS. A constant bias current of +20 nA was applied to the iontophoresis microelectrode from a high-impedance source (model ION-100, Dagan Corp., Minneapolis, MN) to maintain a stable nt
(Nicholson and Phillips, 1981
). To generate a diffusion curve, an additional 100 nA iontophoretic current pulse was applied every 3-5 min for 50 sec. The subtracted ion and reference signals were amplified and low-pass filtered (4 Hz) using a CyberAmp 380 (Axon-CNS, Molecular Devices, Sunnyvale, CA). Diffusion curves were digitized and recorded with a personal computer where they were converted to concentration using the ISM calibration and analyzed with the programs Wanda and Walter (Hrabětová and Nicholson, 2007
). The electrode array was aligned along a transverse axis at a depth of 400 μm in the somatosensory neocortex; because the neocortex is isotropic (Lehmenkühler et al. 1993
), measurement along a single axis suffices to determine α
. and k
Diffusion measurement in brain slices
All recordings were made in somatosensory neocortical layers III - VI (). The methods were similar to those employed in vivo with the following changes. The iontophoretic microelectrode and the ISM were held in separate robotic micromanipulators (model MP 285; Sutter Instrument Co., Novato, CA), with each microelectrode at an angle of 31° from the horizontal plane () and the two electrode tips advanced into either agarose or cortical slice until they were 200 μm deep and 130 μm apart. The iontophoresis current step varied from 30-100 nA.
Figure 2 Diffusion of TMA+ in the somatosensory neocortex (S1) of AQP4+/+ and AQP4-/- mice in vitro. A. A schematic of coronal brain slice with two independent microelectrodes positioned in S1. B. Representative TMA+ diffusion curves in AQP4+/+ (top) and AQP4 (more ...)
Values are given as mean ± SEM. Differences between groups were analyzed with a two-sample equal variance t-test. Values of P < 0.05 were considered significant.