Mice with targeted Slc4a10 gene disruption have small brain ventricles and show reduced neuronal excitability

Abstract

Members of the SLC4 bicarbonate transporter family are involved in solute transport and pH homeostasis. Here we report that disrupting the Slc4a10 gene, which encodes the Na(+)-coupled Cl(-)-HCO(3)(-) exchanger Slc4a10 (NCBE), drastically reduces brain ventricle volume and protects against fatal epileptic seizures in mice. In choroid plexus epithelial cells, Slc4a10 localizes to the basolateral membrane. These cells displayed a diminished recovery from an acid load in KO mice. Slc4a10 also was expressed in neurons. Within the hippocampus, the Slc4a10 protein was abundant in CA3 pyramidal cells. In the CA3 area, propionate-induced intracellular acidification and attenuation of 4-aminopyridine-induced network activity were prolonged in KO mice. Our data indicate that Slc4a10 is involved in the control of neuronal pH and excitability and may contribute to the secretion of cerebrospinal fluid. Hence, Slc4a10 is a promising pharmacological target for the therapy of epilepsy or elevated intracranial pressure.

Fig. 1.

Generation of Slc4a10 KO mice. (A) (Top) Partial genomic structure of the Slc4a10 gene. (Middle) Targeted Slc4a10 locus. Dotted line represents the extent of the homology region of the targeting construct. A neomycin selection cassette flanked by loxP sites (gray arrowheads) was inserted into intron 11. A third loxP site was introduced into intron 12. Two correctly targeted ES cell clones were transiently transfected with a Cre-recombinase expression plasmid. (Bottom) ES cell clones with a Cre-mediated excision of the DNA fragment between the outer loxP sites were subsequently used for the generation of chimeric mice. (B) A membrane protein immunoblot with an Slc4a10 antibody confirmed the absence of Slc4a10 in brain tissue of KO mice. (C) In DAB stainings of sagittal brain sections, Slc4a10 localizes to the choroid plexus, cortex, olfactory bulb, cerebellum, brainstem, and spinal cord, but is not detected in large fiber tracts as the corpus callosum. (D) The specificity of the staining was confirmed by its absence in KO tissue.

Fig. 2.

Deletion of Slc4a10 induces changes in the structure and function of choroid plexus epithelial cells and reduces the volume of brain ventricles. (A) Slc4a10 (green) specifically localizes to the basolateral, but not the apical, membrane stained for the Na⁺/K⁺-ATPase (red) of the choroid plexus epithelium. (B) Specificity of the signal was confirmed by its absence in the choroid plexus tissue from KO mice. (C and D) In T₁-weighted 3D FLASH manganese-enhanced MRI scans, fluid-filled space appears dark and choroid plexus appears white. (E) Volume of brain ventricles was subsequently calculated from WT (n = 3) and KO (n = 4) mouse brains. In KO mice, it was reduced to ≈25% of the WT volume (mean ± SEM). **, P < 0.01. (F and G) Ultrastructural analysis of the KO choroid plexus epithelium (G) revealed enlarged lateral intercellular spaces (*) and a reduction of apical microvilli (arrowheads), compared with WT (F). Small arrows mark lateral cell borders, whereas large arrows mark the basal cell pole. bv, blood vessel. (H) Representative recordings of pH_i from individual cells of choroid plexus from WT and Slc4a10 KO mice. Cells equilibrated in bicarbonate-buffered salt solution (BBS) were acidified by an NH₄Cl pulse, which was washed out by Na⁺-free media (−Na). (I) Rate of Na⁺-dependent pH_i recovery (dpH_i/dt) was measured after readdition of Na⁺ (BBS) at the time of peak acidosis. dpH_i/dt was calculated from the slopes of the respective curves (mean dpH_i/dt ± SEM; WT, n = 6; KO, n = 5). *, P < 0.05. (J) Proposed model for the secretion of CSF. Na⁺ enters the choroid plexus epithelial cells at the basolateral side mainly by Slc4a10. It is secreted apically by Na⁺-bicarbonate cotransport and the Na⁺/K⁺-ATPase. Water follows passively by aquaporin 1 water channels. (Scale bars: A and B, 30 μm; F and G, 1 μm.)

Fig. 3.

Cellular and subcellular localization of Slc4a10 in hippocampus. (A) DAB stainings of the hippocampus reveal that Slc4a10 is abundant in the CA3 region (sagittal). The dashed frames indicate the regions displayed at higher magnifications in C and E. (B) Staining was absent in KO tissue. (D) Some Slc4a10-positive cells were identified as interneurons because of their expression of GAD. (E) In CA3, the stratum oriens, stratum pyramidale, stratum radiatum, and lacunosum moleculare stained positive for Slc4a10. (F–H) Slc4a10 did not localize to axons stained for NF (F) but localized to MAP2-stained dendrites of pyramidal cells (G and H). (I and J) Slc4a10 was often closely associated with GAD, a presynaptic marker of inhibitory interneurons. (K and L) In electron micrographs of DAB-stained sections of the CA3 region, somata (so) (K), dendrites (d) (L), and spines (sp) were stained. (Scale bars: A, 0.5 mm; C and E, 1 mm; D, F, G, and I, 5 μm; K, 500 nm.) Slc4a10 is shown in green; MAP2, NF, and GAD are shown in red; nuclei are stained in blue.

Fig. 4.

An exogenous acid load reveals a defect in pH_i regulation in Slc4a10 KO slices with a parallel in network activity. (A) Grand averages of pH_i responses upon an intracellular acid load induced by bath application of 20 mM propionate for 13 min (horizontal bar) from CA3 stratum pyramidale (12 Slc4a10 KO slices, gray line; 10 WT slices, black line). Upward deflections indicate an increase in pH_i. (B–D) Mean ± SEM values of the peak acid shift in pH_i induced by propionate (B), recovery of pH_i in percentage of peak acidosis by the end of the propionate application (C), and amplitude of the alkaline overshoot during propionate washout (D). *, P < 0.05; **, P < 0.01. (E) Effect of 10 mM propionate on the occurrence of 4-AP-induced interictal-like events in WT (filled circles; n = 20 slices) and Slc4a10 KO slices (open circles; n = 27 slices) shown as a relative mean (+ SEM) frequency calculated from a sliding 3-min time window. (Insets) Sample traces of interictal-like events recorded with field potential electrodes in the hippocampal CA3 pyramidal cell layer under control conditions (Left) and during 25-min propionate application (Right) in the WT (Upper) and Slc4a10-KO (Lower) slices.

Fig. 5.

Slc4a10 KO mice have a higher seizure threshold. (A) After i.p. injection of PTZ, Slc4a10 KO mice did not experience generalized seizures (GEN) (WT, n = 10; KO, n = 9). MJ, myoclonic jerk; CL, clonic seizure. (B) KO mice were protected against fatal epileptic activity after induction of generalized epileptic activity by pilocarpine. (C) Onset of hyperthermia-induced generalized epileptic activity in 10-day-old KO pups was significantly delayed. Shown are means + SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.