Predominant Functional Expression of Kv1.3 by Activated Microglia of the Hippocampus after Status epilepticus

BACKGROUND:Growing evidence indicates that the functional state of microglial cells differs according to the pathological conditions that trigger their activation. In particular, activated microglial cells can express sets of Kv subunits which sustain delayed rectifying potassium currents (Kdr) and modulate differently microglia proliferation and ability to release mediators. We recently reported that hippocampal microglia is in a particular activation state after a status epilepticus (SE) and the present study aimed at identifying which of the Kv channels are functionally expressed by microglia in this model. METHODOLOGY/PRINCIPAL FINDINGS:SE was induced by systemic injection of kainate in CX3CR1(eGFP/+) mice and whole cell recordings of fluorescent microglia were performed in acute hippocampal slices prepared 48 h after SE. Microglia expressed Kdr currents which were characterized by a potential of half-maximal activation near -25 mV, prominent steady-state and cumulative inactivations. Kdr currents were almost abolished by the broad spectrum antagonist 4-Aminopyridine (1 mM). In contrast, tetraethylammonium (TEA) at a concentration of 1 mM, known to block Kv3.1, Kv1.1 and 1.2 subunits, only weakly reduced Kdr currents. However, at a concentration of 5 mM which should also affect Kv1.3 and 1.6, TEA inhibited about 30% of the Kdr conductance. Alpha-dendrotoxin, which selectively inhibits Kv1.1, 1.2 and 1.6, reduced only weakly Kdr currents, indicating that channels formed by homomeric assemblies of these subunits are not important contributors of Kdr currents. Finally, agitoxin-2 and margatoxin strongly inhibited the current. CONCLUSIONS/SIGNIFICANCE:These results indicate that Kv1.3 containing channels predominantly determined Kdr currents in activated microglia after SE

Status epilepticus induces a particular microglial activation state characterized by enhanced purinergic signaling.: Activated microglia in the epileptic hippocampus

International audienceMicroglia cells are the resident macrophages of the CNS, and their activation plays a critical role in inflammatory reactions associated with many brain disorders, including ischemia, Alzheimer's and Parkinson's diseases, and epilepsy. However, the changes of microglia functional properties in epilepsy have rarely been studied. Here, we used a model of status epilepticus (SE) induced by intraperitoneal kainate injections to characterize the properties of microglial cells in hippocampal slices from CX3CR1(eGFP/+) mice. SE induced within 3 h an increased expression of inflammatory mediators in the hippocampus, followed by a modification of microglia morphology, a microglia proliferation, and a significant neurodegeneration in CA1. Changes in electrophysiological intrinsic membrane properties of hippocampal microglia were detected at 24-48 h after SE with, in particular, the appearance of new voltage-activated potassium currents. Consistent with the observation of an upregulation of purinergic receptor mRNAs in the hippocampus, we also provide pharmacological evidence that microglia membrane currents mediated by the activation of P2 receptors, including P2X(7), P2Y(6), and P2Y(12), were increased 48 h after SE. As a functional consequence of this modification of purinergic signaling, motility of microglia processes toward a source of P2Y(12) receptor agonist was twice as fast in the epileptic hippocampus. This study is the first functional description of microglia activation in an in vivo model of inflammation and provides evidence for the existence of a particular microglial activation state after a status epilepticus

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Effects of broad spectrum blockers of potassium channels on the outward rectifying current expressed by microglia 48 h after the induction of <i>status epilepticus</i>.

<p>A Example of the current (left panel) induced by a voltage step from −70 to +30 mV in control (black trace) and after perfusion of 4-AP (1 mM, red trace), and the I/V relationships in the same cell (right panel). Note that 4-AP almost completely abolished the outward rectifying currents without affecting the inward currents. B–C Examples of leak subtracted currents induced by a voltage step from −70 to +40 mV in control (black trace) and after 4-AP 1 mM (B, red trace) or TEA 5 mM (C). The leak conductances of the cells in B and C were 585 and 256 pS, respecitively. The graphs on the right represent the conductance, normalized to its maximum value, as a function of membrane potential and its inhibition induced by 4-AP (B, n = 8), TEA (C) 1 mM (n = 9) and 5 mM (n = 7).</p

Summary of the effects induced by the different drugs tested on the outward rectifying potassium current evoked by a voltage step from −70 to +30 mV.

<p>The histogram represents the average of the leak subtracted current after drug application and normalized to its pre-drug value. The “time matched control” bar corresponds to experiments in which the current was measured during 10 to 15 minutes without any drug application to control for the absence of any significant run-down of the current. Statistical tests were done on raw data (paired <i>t test</i>, **p<0.01; the number of tested cells for each condition is that given in the legends of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006770#pone-0006770-g003" target="_blank">figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006770#pone-0006770-g004" target="_blank">4</a>).</p

Biophysical properties of potassium channels expressed in microglial cells after <i>status epilepticus</i>.

<p>A1 Example of currents used to obtain activation and inactivation curves in microglia after <i>status epilepticus</i>. Microglia cells were held at a potential of −70 mV and stepped at different potentials for 1 s before a final step of 250 ms at +50 mV. The inset represents the voltage steps corresponding to the current traces showed in the figure. A2 Average of activation and inactivation curves (n = 13) normalized to maximal conductance. B1 Examples of currents induced in another microglial cell by 10 consecutive pulses from −70 to +30 mV with 30 s interval (left panel) or 0.4 s interval (right panel). B2 Evolution of the peak current amplitude obtained at every pulse expressed as percentage of the first response for 7 tested cells. Note that the smaller is the inter-pulse interval, the higher is the inactivation of the current.</p

Calcium-activated potassium currents in resting and activated microglia.

<p>A, B Examples of currents induced by voltage steps from 0 to +80 mV before (black traces) and after (red traces) bath application of TEA (1 mM) with 1 µM (left panel) and 0 µM (right panel) estimated intracellular free calcium in microglial cells from control (A) and from epileptic (B) mice. C Summary of the effects of TEA (1 mM, red column) and intracellular calcium on the current densities induced by voltage steps from 0 to +80 mV in microglia of control and epileptic mice (paired <i>t test</i>, *p<0.05).</p

Effects of α-dendrotoxin (A, 50 nM), agitoxin-2 (B, 10 and 50 nM) and margatoxin (C, 1 and 10 nM) on the leak subtracted current induced by a voltage step from −70 to +40 mV (black traces recorded in control, red traces after drug application).

<p>The leak conductances of the cells in A, B and C were 338, 862 and 332 pS, respectively. The graphs on the right represent the conductance, normalized to its maximum value, as a function of the membrane potential and its inhibition by α-dendrotoxin (A, n = 4, Dtx), agitoxin-2 (B, AgTx, n = 14 for 10 nM, n = 6 for 50 nM) and margatoxin (C, MgTX, n = 8 for 1 nM, n = 11 for 10 nM).</p