Jacobs et al. 10.1073/pnas.0705487105. |
Fig. 6. Survival and weight gain of Slc4a10 KO mice. (A) In mixed litters on a regular diet, »80% (27 of 34) of Slc4a10 KO mice (gray) died before 60 days of age, whereas only a few WT mice died (2 of 35, black bars). If soft food was offered ±2 weeks around weaning, the majority of KO mice survived. (B) In mixed litters, KO mice (n = 22) did not gain bodyweight at the same rate as their WT littermates (n = 35), even if fed with soft food, but finally reached a normal bodyweight. (C) The weight gain of KO progeny from surviving KO mice fed with regular food growing up in small litters did not differ from that of WT mice (n = 8 for each genotype).
Fig. 7. Initial characterization of Slc4a10 KO mice. (A) In a multiple-tissue Northern blot from organs of adult WT mice hybridized with a Slc4a10-cDNA probe, transcripts were detected in brain, retina, and spinal cord. (B) Northern analysis of wild-type (+/+), heterozygous (+/-), and KO (-/-) brain tissue with this probe revealed no detectable aberrant transcripts in KO tissue. (C) Transcript abundance assayed by using quantitative real-time PCR normalized to Hprt shown as x-fold increase, compared with WT for members of the Slc4, Slc9, Slc12, and Slc26 families, which are known to transport chloride, bicarbonate, or protons, is not altered in Slc4a10 KO mice (black, WT, gray, KO).
Fig. 8. Slc4a10 KO mice habituate more slowly to novel stimuli, but they do not show clear deficits in motor coordination or spatial learning. (A and B) Overall activity was determined by recording mouse movements for 11 days. Compared with WT mice (n = 6) (A), mean activity and the diurnal rhythm of locomotion were unchanged in Slc4a10 KO mice (n = 4) (B). (C) KO (n = 10) and control (n = 11) mice were placed individually on an accelerating rotating rod three times daily on 2 consecutive days, and the mean performance was evaluated for each trial. The numbers at the bottom indicate the individual training session. No major difference was observed between the genotypes. (D) KO (n = 13) and WT mice (n = 15) were placed into a brightly lit cage with a dark house. The time spent in the dark or bright area and during the transitions between the bright and dark area was measured for a 10-min period. The time that KO mice spent in the bright part of the cage was increased, compared with WT mice. (E and F) The decrease in locomotor activity in the open field during successive 5-min sessions was delayed in KO mice, compared with WT mice on the first and second days (data not shown), whereas the difference was gone on the third day (n = 13 per genotype). (G) The time that mice needed to find the hidden platform in the Morris water maze was recorded in 12 trials per day on 3 consecutive days and was not prolonged in the KO versus WT animals (KO, n = 13; WT, n = 12). The averages of four consecutive experiments are shown. (H) When the platform was removed on the fourth day, mice of both genotypes preferentially swam in the quadrant where the platform had been located. Shown are mean ± SEM. *, P < 0.05.
Fig. 9. Expression of Slc4a10. As shown for the CA3 region of the hippocampus, Slc4a10 neither colocalizes with CNPase (A-C′), a marker for oligodendrocytes, nor with GFAP (D-F′), a marker of astrocytes. (G) In Western blots, Slc4a10 was detected in protein lysates of mixed glia/neuron cultures, but not in glia cell cultures. (Scale bars: A and D, 5 mm.)
Fig. 10. Localization of Slc4a10 in neocortex and cerebellum. (A-C) Slc4a10 is broadly expressed in the neocortex and colocalizes with some interneurons (arrow) labeled by GAD. (D-F) In the cerebellum, Slc4a10 is abundant in Purkinje cells (pc) that were labeled by parvalbumine, but was not found in interneurons of the molecular layer (ml). (Scale bars: A and D, 10 mm.)
Fig. 11. MRI analysis. ADC maps, T2 maps, and T2-weighted (T2w) MR images (symbol) of the brains of Slc4a10 KO and control mice are shown. Note how the lateral and third ventricles (white arrows) are barely visible in KO mice.
Fig. 12. Histological analysis of the choroid plexus and the hippocampal region of 6-month-old Slc4a10 KO mice. (A and B) Methylen blue stainings show a free-floating choroid plexus in WT mice, whereas it touches the walls of the ventricle in KO mice. (C and D) Nissl-and (E and F) H&E stainings of the hippocampus did not reveal overt morphological changes in KO brain tissue. (Scale bars: A and C, 200 mm; E, 30 mmin.)
Table 1. Quantification of MRI data
Region of interest | Slc4a10 KO | Slc4a10 WT | D | P |
T2, ms | ||||
WHB | 0.069 ± 0.001 | 0.075 ± 0.001 | -8.1% | 0.034 |
COR | 0.067 ± 0.002 | 0.070 ± 0.001 | -3.8% | 0.212 |
CC | 0.066 ± 0.001 | 0.067 ± 0.001 | -1.1% | 0.589 |
HIP | 0.068 ± 0.002 | 0.074 ± 0.002 | -8.2% | 0.077 |
THA | 0.064 ± 0.001 | 0.066 ± 0.001 | -2.8% | 0.138 |
AMY | 0.072 ± 0.000 | 0.073 ± 0.001 | -0.9% | 0.554 |
MSC | 0.042 ± 0.001 | 0.038 ± 0.001 | +9.8% | 0.048 |
ADC, mm2/s | ||||
WHB | 0.450 ± 0.016 | 0.518 ± 0.013 | -13.1% | 0.074 |
COR | 0.485 ± 0.010 | 0.503 ± 0.016 | -3.5% | 0.285 |
CC | 0.374 ± 0.014 | 0.353 ± 0.022 | +6.2% | 0.480 |
HIP | 0.414 ± 0.022 | 0.522 ± 0.012 | -20.7% | 0.034 |
THA | 0.437 ± 0.036 | 0.450 ± 0.010 | -2.9% | 1.000 |
AMY | 0.456 ± 0.006 | 0.523 ± 0.013 | -12.7% | 0.034 |
MSC | 0.889 ± 0.066 | 01.021 ± 0.044 | -12.9% | 0.157 |
T2 relaxation times (T2) and apparent diffusion coefficients (ADC) in Slc4a10 KO and WT mice are given. Results are expressed as mean ± SEM. Difference (Δ) between KO and WT mice is expressed in percentages. Statistical significance (P) was calculated by using a nonparametric Mann-Whitney U test. WHB, whole brain; COR, cerebral cortex; CC, corpus callosum; HIP, hippocampus; THA, thalamus; AMY, amygdala; MSC, muscle.
SI Methods
Generation of Slc4a10 KO Mice.
A clone isolated from a 129/SvJ mouse genomic l library (Stratagene) was used to construct the targeting vector. An 8-kb fragment including exons 11 to 14 of the Slc4a10 gene was cloned into the pKO-V901 plasmid (Lexicon Genetics) with a phosphoglycerate kinase (pgk) promoter-driven diphtheria toxin A cassette. A pgk promoter-driven neomycin resistance cassette flanked by loxP-sites was inserted into the MunI-site in intron 11. A third loxP-site and an additional SpeI-site were inserted into the SwaI-site in intron 13. The construct was electroporated into R1 mouse ES cells. Neomycin-resistant clones were analyzed by Southern blot by using SpeI and an external ~500-bp probe. Correctly targeted ES cells were transfected with a plasmid-expressing Cre-recombinase to remove the neomycin cassette and exon 12. Correctly recombined clones were identified with an internal probe by Southern blot analysis. Two independent ES cell clones were injected into C57B/L6 blastocysts to generate chimeras that were backcrossed with C57B/L6. Studies were performed in a mixed 129SV/C57Bl6 background in the F4 and F5 generations. We used littermates as controls. Genotypes were either determined by Southern blot or PCR on tail biopsy DNA. For PCR genotyping, the sense primer F1 (CTGCAAGCAATGTGTGAGGAG) and the antisense primers R1 (CTCCCTACAGACCTCCAACAGCG) and R2 (GAGCAGCCCAGATGTACACCAGC) were used in a single PCR mix. The primer pair F1/R1 amplified a 694-bp knockout allele, and the primer pair F1/R2 amplified a 598-bp WT allele.Behavioral Analysis.
For the rotarod assay, mice were placed on a rotating rod (TSE Systems), and the time spent on the rotarod was measured for a period of up to 180 s. Three trials per day were performed on two consecutive days (KO, n = 10; WT, n = 11). For the activity assay, the home cage activity of single mice (KO, n = 4; WT, n = 6) was monitored for 11 days with an infrared motion detector with a sampling frequency of 1 Hz and a bin size of 2 min. For the open field assay, mice were placed in 50 ´ 50-cm box (n = 13 per genotype) for 25 min for 3 consecutive days. The movement of the mice and the time spent in the center of the box were recorded by a video system (TSE VideoMot2; TSE Systems). To observe the exploration of a novel object, a clean 50-ml falcon tube was placed in the center of the box (n = 13 per genotype) after the last open field recording period.The testing apparatus for the dark-light assay was divided into a bright, open-topped and a dark, closed-topped compartment connected by a door. Each mouse was placed in the doorway facing the dark compartment (KO, n = 13; WT, n = 15). The time spent outside the dark area and the transitions between the bright and dark areas were measured for 10 min by using the TSE VideoMot 2 system.
For the Morris water maze, a circular swimming pool of 150-cm diameter was filled with opaque water. It was located in a room with various distal cues. Inside the pool a platform (13 cm in diameter) was positioned such that its top surface was 0.5 cm below the surface of the water. Data were collected by using a video camera fixed to the ceiling. The time the mice needed to find the hidden platform was recorded for 12 trials per day on 3 consecutive days. On the fourth day, the platform was removed, and the covered distance and the time spent in the platform-quarter of the mice were recorded in four consecutive sessions, each starting from a different quadrant (KO, n = 13; WT, n = 12).
Morphology.
Adult mice were deeply anesthetized and perfused transcardially with 0.1 M phosphate buffer [(PB) 77.4 mM Na2HPO4, 22.6 mM NaH2PO4 (pH 7.4)], followed by 4% paraformaldehyde in PB for 5 min. Organs were removed, postfixed overnight, subsequently dehydrated in an ascending isopropanol series, and paraffin-embedded. Then 10-mm sections were cut, deparaffinized, and H/E, methylenblue, or Nissl stained by using standard procedures.For immunohistochemistry the brain was removed after transcardial perfusion and postfixed in 4% paraformaldehyde in 0.1 M PB for 1 h. The tissue was left in 30% sucrose overnight. Staining was done on free-floating 50-mm cryosections as described elsewhere. Primary antibodies were from Roche (MAP2), the Developmental Studies Hybridoma Bank (Iowa) (NF), and Alexis (GAD). Secondary antibodies were the Alexa Fluor 546 coupled goat anti-rabbit and Alexa Fluor 488 coupled goat anti-mouse (Molecular Probes). Analysis was done by confocal microscopy (Leica SP2). For DAB staining, sections were incubated with a biotinylated anti-rabbit IgG secondary antibody by using the vectastain ABC-kit (Vector). The peroxidase stain was visualized by 0.05% 3,3-diaminobenzidine, 0.04% nickel ammonium sulfate, and 0.03% H2O2 dissolved in 0.01 M PB. For EM, mice were perfused with 4% PFA and 0.5% glutaraldehyde in 0.1 M PB. Tissue was cut into 50-mm sections with a vibrating blade microtome (VT 1000S; Leica) and stained as described earlier. After fixation with 1% osmiumtetroxide, the slides were dehydrated in an ascending series of ethanol and embedded in Epon. Ultrathin sections were examined with a Zeiss EM 902.
Seizure Susceptibility.
PTZ (Sigma-Aldrich) dissolved in PBS was administered i.p. at two different doses [40 and 60 mg/kg bodyweight (bw)] in a total volume of 200 and 300 ml, respectively. After injection, the animals were watched closely for 10 min. The latency until the first myoclonic jerk (focal seizure), clonic seizure (clamping of the forefeet), or generalized (tonic/clonic) seizures was measured. Pilocarpine (Sigma-Aldrich) was dissolved in PBS and injected i.p. at a dose of 350 mg/kg bw (volume 300 ml). Then 30 min before pilocarpine treatment, mice received 1 mg/kg bw methyl-scopolamine i.p. (Sigma-Aldrich) dissolved in PBS (total volume 100 ml) to block peripheral cholinergic actions of pilocarpine. For hyperthermia-induced epileptic activity, 10-day-old pups of both genotypes of a comparable bw were placed into a chamber with an ambient temperature of 48.0 ± 1°C (1). The latency until loss of postural control because of sustained tonic/clonic generalized epileptic activity (>20 s) was taken.Recording of pHi of Choroid Plexus Cells.
To detach epithelial cells, mouse choroid plexi were treated for 15 min with 4 mg/ml dispase and 1 mg/ml collagenase A in 37°C BBS [142 mM Na+, 0.8 mM Mg2+, 0.8 mM SO42-, 111.6 mM Cl-, 4.0 mM K+, 1.8 mM Ca2+, 24.0 mM HCO3-, 14.0 mM Hepes, 5.6 mM glucose (pH 7.4), and bubbled with 5% CO2 in atmospheric air]. The cells were mounted in a closed perfusion chamber (358 ml vol RC-21BR; Harvard Apparatus), loaded for 10-15 min with 0.1 mM BCECF-AM, and superfused with a linear flow rate of 0.8 mm/s corresponding to 1 ml/min or 2.8 bath changes per min during recording.The excitation ratio (495 nm/440 nm) of the 510- to 535-nm light emission was recorded and calibrated as described before (2). Cells were acidified by NH4Cl prepulse (3-5 min; BBS with equimolar substitution of Na+ by NH4+), shifted to a Na+-free buffer for 1-2 min (substitution of Na+ by N-methyl-D-Glucamine), and then shifted back to BBS. The rate of pHi recovery (dpHi/dt; data from three individual cells per recording and one to six recordings per animal pooled for each experiment) over 12 s was determined after reintroduction of Na+, and a two-tailed Mann-Whitney rank sum test was used for statistical analysis.
Electrophysiology and pHi Fluorescence Imaging in Hippocampal Slices.
Horizontal hippocampal slices (400 mm) from 8- to 12-week-old mice were prepared (recovery at room temperature for at least 1 h), and field potential recordings from CA3 stratum pyramidale were done as described (3). An equimolar amount of D-gluconate was substituted for Cl- in the control solution [124 mM NaCl, 3.0 mM KCl, 2.0 mM CaCl2, 25 mM NaHCO3, 1.1 mM NaH2PO4, 2 mM MgSO4, and 10 mM D-glucose, and bubbled with 95% O2, 5% CO2 (pH 7.4) at 32°C] in experiments where propionate was used to evoke pHi changes. The detection threshold for interictal-like events induced by 4-AP (Fluka) was at least 4 SD of the baseline noise amplitude. For pHi measurements, 200-mm slices were incubated with 5 mM BCECF-AM (Molecular Probes) for ~25 min and washed for 20-40 min in control solution. In each slice pHi was measured as described (4) from 17-29 CA3 pyramidal neurons (32-33°C). The pH signals from individual cells were averaged in a slice before averaging across slices.MRI Analysis.
Slc4a10 KO (n = 4) and control (n = 3) mice were imaged by using a 4.7T scanner at the age of P153 ± 23. Mice were anesthetized by using 75 mg/kg ketamine and 1 mg/kg medetomidine. A bolus of manganese chloride (50 mg/kg) was injected into the tail vein just before imaging. T2-weighted scout images were taken in axial, coronal, and sagittal orientations. High-resolution T1-weighted 3D FLASH-imaging sequences [repetition time = 50 ms, echo time = 7, flip angle = 25°, averages = 4, resolution = (150 mm)3, acquisition time = 30 min 43 s] were acquired according to the scout images. In a second set of mice, T2 and ADC maps were acquired (Slc4a10-KO, n = 3; control, n = 4). T2 maps were recorded by using a multispin echo sequence (repetition time = 4,500 ms, echo time = 15 ms - 180 ms, 12 echoes, averages = 2, matrix = 128 ´128, field of view = 2.0 cm ´ 2.0 cm, 1 coronal slice, acquisition time = 19 min 12 s). ADC maps were recorded by using a spin echo sequence (repetition time = 2,000 ms, echo time = 30 ms, D = 20 ms, B0 = 18 s/mm2, B1 = 996 s/mm2, gradient strengths 0 and 157 for B values, respectively, d = 5 ms, averages = 3, matrix = 128 ´ 128, field of view = 2.0 cm ´ 2.0 cm, 1 coronal slice, acquisition time = 25 min 36 s). 3D image analysis was performed by using MRIcro program (version 1.39 build 4). A region-of-interest (ROI) was drawn on manganese-enhanced choroid plexus, and the volume of this ROI was measured by multiplying the number of the volume elements with the volume of one such element. Student's t test was used for testing statistical significance. T2 and ADC maps were generated by using ImageJ program (version 1.35c; National Institutes of Health) with MRI analysis calculator (version 1.0) developed by Karl Schmidt. The average T2 and ADC values within each ROI were measured, and statistical significance of the changes were tested by using a Mann-Whitney U test in SPSS software (version 15.0; SPSS). All numerical data are expressed as mean ±SEM.1. Schuchmann S, Schmitz D, Rivera C, Vanhatalo S, Salmen B, Mackie K, Sipila ST, Voipio J, Kaila K (2006) Nature Med 12:817-823.
2. Bouzinova EV, Praetorius J, Virkki LV, Nielsen S, Boron WF, Aalkjaer C (2005) Am J Physiol Cell Physiol 289:C1448-C1456.
3. Sipilä ST, Huttu K, Soltesz I, Voipio J, Kaila K (2005) J Neurosci 25:5280-5289.
4. Ruusuvuori E, Li H, Huttu K, Palva JM, Smirnov S, Rivera C, Kaila K, Voipio J (2004) J Neurosci 24:2699-2707.