Conditional knock-out of Kir4.1 leads to glial membrane depolarization, inhibition of potassium and glutamate uptake, and enhanced short-term synaptic potentiation

Abstract

During neuronal activity, extracellular potassium concentration ([K+]out) becomes elevated and, if uncorrected, causes neuronal depolarization, hyperexcitability, and seizures. Clearance of K+ from the extracellular space, termed K+ spatial buffering, is considered to be an important function of astrocytes. Results from a number of studies suggest that maintenance of [K+]out by astrocytes is mediated by K+ uptake through the inward-rectifying Kir4.1 channels. To study the role of this channel in astrocyte physiology and neuronal excitability, we generated a conditional knock-out (cKO) of Kir4.1 directed to astrocytes via the human glial fibrillary acidic protein promoter gfa2. Kir4.1 cKO mice die prematurely and display severe ataxia and stress-induced seizures. Electrophysiological recordings revealed severe depolarization of both passive astrocytes and complex glia in Kir4.1 cKO hippocampal slices. Complex cell depolarization appears to be a direct consequence of Kir4.1 removal, whereas passive astrocyte depolarization seems to arise from an indirect developmental process. Furthermore, we observed a significant loss of complex glia, suggestive of a role for Kir4.1 in astrocyte development. Kir4.1 cKO passive astrocytes displayed a marked impairment of both K+ and glutamate uptake. Surprisingly, membrane and action potential properties of CA1 pyramidal neurons, as well as basal synaptic transmission in the CA1 stratum radiatum appeared unaffected, whereas spontaneous neuronal activity was reduced in the Kir4.1 cKO. However, high-frequency stimulation revealed greatly elevated posttetanic potentiation and short-term potentiation in Kir4.1 cKO hippocampus. Our findings implicate a role for glial Kir4.1 channel subunit in the modulation of synaptic strength.

Figure 1.

Generation of K_ir4.1 cKO mice. A, Targeting construct for the recombinant K_ir4.1^f/f mouse line. neo/tk selection cassette was removed by crossing K_ir4.1^f/f and FLPeR mice. LA, Left arm homology; RA, right arm homology. B, Construct for the transgenic hGFAP–Cre line. mP1, Poly(A) signal from mouse protamine1 gene. C, Kir4.1 Western blot from P20 littermate WT, K_ir4.1^f/f, K_ir4.1^+/−, and K_ir4.1 cKO brain and kidney tissue. Blotting for β-actin was used as a loading control.

Figure 2.

K_ir4.1 cKO phenotype. K_ir4.1 cKO mice exhibit ataxia and hindleg paralysis (A), stress-induced seizures (B), growth retardation (C), and premature lethality (D).

Figure 3.

Loss of K_ir4.1 from the CNS gray and white matter. A, Fluorescent GFP and K_ir4.1 immunostaining in WT/S100β–eGFP and K_ir4.1 cKO/S100β–eGFP hippocampus. DG, Dentate gyrus; CA1, CA1 pyramidal cell layer. Scale bar, 50 μm. B, C, Fluorescent immunostaining for K_ir4.1, astrocyte marker GLAST (B) and oligodendrocyte marker CNP (C) in WT and K_ir4.1 cKO cerebellum. GL, Granule cell layer; ML, molecular layer; WM, white matter. Scale bar, 500 μm. D, Fluorescent immunostaining for K_ir4.1 and astrocyte marker GLAST in WT and K_ir4.1 cKO spinal cord. Scale bar, 100 μm.

Figure 4.

Morphological changes and white-matter vacuolization in the K_ir4.1 cKO CNS. A, Paraffin-embedded sagittal sections of the WT and K_ir4.1 cKO cerebellum stained with solochrome and eosin. Scale bar, 1 mm. B, Left, Transverse thoracic spinal cord sections stained with solochrome and eosin. Scale bar, 200 μm. Right, Longitudinal thoracic spinal cord sections stained with hematoxylin and eosin. Scale bar, 50 μm. C, Sagittal sections of the hippocampus stained with hematoxylin and eosin. Scale bar, 500 μm.

Figure 5.

Membrane properties of the hippocampal passive and complex glia. A, Representative whole-cell currents of WT and K_ir4.1 cKO complex glia in CA1 stratum radiatum. Middle trace depicts Ba²⁺ block of whole-cell currents in a WT complex cell. B, R_m and V_m of complex glia. Mean ± SEM; *p < 0.05. C, Representative whole-cell currents of WT and K_ir4.1 cKO passive glial cells in CA1 stratum radiatum. Middle trace depicts Ba²⁺ block of whole-cell currents in a passive WT cell. D, R_m and V_m of passive glia. Mean ± SEM; *p < 0.05.

Figure 6.

Potassium and glutamate uptake by astrocytes. A, Whole-cell current traces of WT and K_ir4.1 cKO passive astrocytes during Schaffer collateral stimulation in control condition (black trace), after Ba²⁺ block (dark gray trace), and after Ba²⁺/TBOA block (light gray trace). Peak amplitude of K⁺ uptake (Ba²⁺-sensitive) current (B) and peak amplitude of GluT (TBOA-sensitive) current (C) in WT and cKO cells. Mean ± SEM; *p < 0.05.

Figure 7.

Spontaneous activity, membrane, and action potential properties of wild-type and K_ir4.1 cKO pyramidal neurons. A, Representative whole-cell currents of WT and K_ir4.1 cKO CA1 pyramidal neurons. B, Influence of the stimulation intensity on the action potential frequency in WT and K_ir4.1 cKO neurons. Mean ± SEM. C, Representative sEPSC recordings. D, sEPSC frequency and peak amplitude in WT and K_ir4.1 cKO CA1 pyramidal neurons. Mean ± SEM; *p < 0.05.

Figure 8.

Synaptic transmission and plasticity in the wild-type and K_ir4.1 cKO hippocampus. A, Representative WT and K_ir4.1 cKO fEPSPs. B, Input/output curve: influence of stimulation intensity on fEPSP slope. C, Paired-pulse facilitation (representative trace from WT shown as inset). D, Long-term potentiation in WT and K_ir4.1 cKO hippocampus.