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. 2006 May 17;26(20):5438-47.
doi: 10.1523/JNEUROSCI.0037-06.2006.

The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus

Anke Wallraff  1 , Rüdiger Köhling, Uwe Heinemann, Martin Theis, Klaus Willecke, Christian Steinhäuser
Affiliations

The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus

Anke Wallraff et al. J Neurosci. .

Abstract

Astrocytic gap junctions have been suggested to contribute to spatial buffering of potassium in the brain. Direct evidence has been difficult to gather because of the lack of astrocyte-specific gap junction blockers. We obtained mice with coupling-deficient astrocytes by crossing conditional connexin43-deficient mice with connexin30(-/-) mice. Similar to wild-type astrocytes, genetically uncoupled hippocampal astrocytes displayed negative resting membrane potentials, time- and voltage-independent whole-cell currents, and typical astrocyte morphologies. Astrocyte densities were also unchanged. Using potassium-selective microelectrodes, we assessed changes in potassium buffering in hippocampal slices of mice with coupling-deficient astrocytes. We demonstrate that astrocytic gap junctions accelerate potassium clearance, limit potassium accumulation during synchronized neuronal firing, and aid in radial potassium relocation in the stratum lacunosum moleculare. Furthermore, slices of mice with coupling-deficient astrocytes displayed a reduced threshold for the generation of epileptiform events. However, it was evident that radial relocation of potassium in the stratum radiatum was not dependent on gap junctional coupling. We suggest that the perpendicular array of individual astrocytes in the stratum radiatum makes these cells ideally suited for spatial buffering of potassium released by pyramidal cells, independent of gap junctions. In general, a surprisingly large capacity for K+ clearance was conserved in mice with coupling-deficient astrocytes, indicating that gap junction-dependent processes only partially account for K+ buffering in the hippocampus.

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Figures

Figure 1.
Figure 1.
Deletion of Cx30 in addition to Cx43 (dko) in hippocampal astrocytes abolishes tracer coupling. A, Tracer-coupled astrocytes in a wt hippocampal slice, as obtained after biocytin diffusion from a single cell, held in whole-cell voltage clamp for 20 min. B and C display the reduced numbers of tracer-coupled astrocytes in hippocampal slices of a Cx43fl/fl:hGFAP-Cre mouse and a Cx30+/−, Cx43fl/fl:hGFAP-Cre mouse, respectively. D, Absence of tracer coupling in a hippocampal slice of a Cx30−/−, Cx43fl/fl:hGFAP-Cre (dko) mouse. Note that, with strongly reduced (C) or abolished (D) tracer coupling, fine astrocytic processes become visible. Scale bar, 50 μm. E, Scheme for quantification of tracer coupling. Slices, 60 μm thick, obtained from a 300 μm slice, were processed for biocytin visualization. In the top three slices, all biocytin-positive cells were counted. F, Summary of the amount of tracer coupling in the different genotypes. *p < 0.5. Error bars represent SEM. s.p., Stratum pyramidale; s.r., stratum radiatum; s.l.m., stratum lacunosum moleculare.
Figure 2.
Figure 2.
dko astrocytes and wt astrocytes share similar morphology and whole-cell currents. A, Tracer-filled astrocyte in a wt hippocampal slice. Low bicarbonate bath solution, pH 6.4, was applied 30 min before and during whole-cell recordings (20 min) to block gap junctional coupling. Under these conditions, this cell was tracer coupled only with two other astrocytes. Note the prominent primary processes and the dense net of fine processes. The arrow indicates a blood vessel encircled by an astrocytic end foot. Scale bar, 50 μm. The inset represents current responses elicited by 50 ms voltage steps from −180 to 0 mV (Vhold of −90 mV). Calibration: 10 ms, 4 nA. B–D represent examples of tracer-filled astrocytes in slices of dko mice, using standard ACSF. Arrows denote astrocytic pericapillary end feet. Insets and calibration as in A. Note that the astrocyte in D displays a bipolar morphology with an orientation perpendicular to the stratum pyramidale (s.p.). E and F depict immunofluorescent anti-GFAP stainings of the stratum radiatum of wt and dko mice, respectively. The insets show stainings of the stratum lacunosum moleculare. Densities of GFAP-positive cells were similar in wt and dko mice, each with a higher density in stratum lacunosum moleculare compared with the stratum radiatum. Note that major processes of stratum radiatum astrocytes often were oriented perpendicularly to the stratum pyramidale. Scale bar, 50 μm.
Figure 3.
Figure 3.
Gap junctions account for ∼30% of whole-cell currents in wt astrocytes. A illustrates the blocking action of l.b./l.pH bath solution, pH 6.4, on tracer coupling in a wt hippocampal slice (30 min preapplication, 20 min recordings). Scale bar, 50 μm. B and C demonstrate the effect of l.b./l.pH solution on whole-cell currents in wt and dko astrocytes, respectively. The insets show representative examples (voltage steps as in Fig. 2); current–voltage relationships in control ACSF (black circles) and l.b./l.pH solution (gray circles) represent means and SEM of five cells. Current amplitudes were normalized to the amplitude recorded at 0 mV. Calibration: 1 nA, 10 ms. D, The amount of bicarbonate-sensitive currents in wt (white bars; 53 ± 5% at −20 mV and 45 ± 5% at −160 mV; n = 5) and dko (dark gray bars; 22 ± 3% at −20 mV and 18 ± 3% at −160 mV; n = 5) mice. For every holding potential, current amplitude in standard ACSF was considered 100%. Black bars represent the extrapolated amount of gap junction-mediated current in wt astrocytes.
Figure 4.
Figure 4.
Elevated stimulation-induced [K+]o levels in dko mice. A, Experimental setup for [K+]o measurements. The stimulation electrode (stim.) was placed in the alveus (a.), and the recording electrode (rec.) was placed in the CA1 stratum pyramidale (s.p.). B, Typical examples of rises in [K+]o elicited by three paired pulses (0.1 ms, 50 ms interval, 30 s interspike interval) at 100% stimulation intensity. Left, wt; right, dko. Scale bar, 5 s. C, Typical examples of rises in [K+]o elicited by trains of stimuli (10 s, 20 Hz) at 25, 50, 75, and 100% stimulation intensity, respectively. Left, wt; right, dko. Scale bar, 5 s. Note that, at maximal stimulation intensity, [K+]o reaches a higher level in dko compared with wt mice. D, Summary of evoked [K+]o rises during double-pulse stimulation. Mean and SEM values of responses to three double pulses were calculated and normalized to amplitudes of concomitant population spikes. White bars, wt; gray bars, dko. Asterisks indicate significant differences (p = 0.015). E, Summary of rises in [K+]o evoked through trains of stimuli, normalized to amplitudes of respective population spikes. White bars, wt; gray bars, dko. Bars represent mean and SEM, and asterisks indicate significant differences (p = 0.001). Note that only wt mice showed a decrease in variation with increasing stimulation strength.
Figure 5.
Figure 5.
Recovery of stimulation-induced increase of [K+]o in stratum pyramidale is slower in dko mice. A, Decline of representative, matched [K+]o transients after stimulus trains (20 Hz) evoked with high (wt, 100%; dko, 75%) and low (wt and dko, 50%) stimulus intensity. For the wt traces, t1/e (the time after which [K+]o amplitude has decayed to 1/e of its initial value) is indicated. Note that decay is faster for larger rises in [K+]o (dotted line, t1/e = 1.93 s; dashed line, t1/e = 2.35 s). B, t1/e values of [K+]o recovery after a stimulus train (20 Hz; 25, 50, 75, and 100% stimulation intensities) plotted against [K+]o at the end of the stimulus (wt, open diamonds; dko, filled squares). The inverse relationships between [K+]o and t1/e were best described by a power function (thin line for wt, y = 5.272x exp(−0.417); thick line for dko mice, y = 6.068x exp(−0.412). y-axis intercepts differed significantly (asterisk; see Results). C, Semilogarithmic plots of the decline of representative, matched [K+]o transients (from 90% to the trough of the undershoot; gray dots) after 100% stimulus trains (20 Hz). Top, wt; bottom, dko. Two exponential components contribute to this decline and are plotted individually (straight black lines; wt, τfast = 1.46 s, τslow = 11.3 s; dko, τfast = 1.15 s, τslow = 10.5 s). Their sum provides an excellent fit of the data (black curves). Note that the relative amplitude of τfast (τfast amplitude fraction) is larger in the wt (84%) compared with the dko [K+]o decay (70%). D, τfast amplitude fractions plotted against [K+]o at the end of the stimulus (wt, open diamonds; dko, filled squares). The linear relationship was significantly (asterisk) shifted toward smaller τfast amplitude fractions in dko mice (thin line, wt, linear equation: y = 0.0445x + 0.39; thick line, dko, y = 0.0346x + 0.38).
Figure 6.
Figure 6.
Impaired spatial buffering in the stratum lacunosum moleculare of dko mice. A, Experimental setup for the analysis of laminar profiles of changes in [K+]o. Stimulation was performed in the alveus (20 Hz, 100%) while the recording electrode was stepped from the stratum pyramidale to the hippocampal fissure (100 μm step size). B, Mean rises in [K+]o (normalized to rise at the stratum pyramidale) plotted against the distance from stratum pyramidale (thin line, dko, n = 8 animals, 15 slices; thick line, wt, n = 8 animals, 13 slices). Normalized [K+]o in dko mice reached lower levels at 400 and 500 μm from the stratum pyramidale compared with wt. For relative changes, see inset. C demonstrates the difference in astrocyte morphology and orientation in the stratum radiatum versus stratum lacunosum moleculare, as obtained after biocytin injection into a stratum radiatum astrocyte proximal to the stratum lacunosum moleculare. Note the small size and random orientation of cells in the stratum lacunosum moleculare. Scale bar, 50 μm. alv., Alveus; fis., fissure; stim., stimulation electrode; rec. recording electrode; s.r. stratum radiatum; s.l.m., stratum lacunosum moleculare.
Figure 7.
Figure 7.
Epileptiform field potentials (EFP) in CA1 stratum pyramidale of dko mice. A, Spontaneous epileptiform field potentials (CA1, stratum pyramidale) occurred only in slices from dko mice (representative trace) but not in slices from wt mice (data not shown; p = 0.001). B, Low-intensity Schaffer-collateral stimulation gave rise to EFP only in dko slices (representative sample trace) but not in slices from wt mice (data not shown) (p = 0.007). C, EFP induced by washout of Mg2+ (0 Mg2+) were more frequent in dko slices and occurred with shorter latency. Original traces of a wt (top) and dko (bottom) slice.
See this image and copyright information in PMC

Comment in

  • Passing potassium with and without gap junctions.
    Kozoriz MG, Bates DC, Bond SR, Lai CP, Moniz DM. Kozoriz MG, et al. J Neurosci. 2006 Aug 2;26(31):8023-4. doi: 10.1523/jneurosci.2500-06.2006. J Neurosci. 2006. PMID: 16888836 Free PMC article. No abstract available.

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