Increasing small conductance Ca2+-activated potassium channel activity reverses ischemia-induced impairment of long-term potentiation

Abstract

Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca(2+)-activated potassium channels type 2 (SK2), expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we showed that, in mice, hippocampal long-term potentiation (LTP) was impaired as early as 3 h after recovery from CA/CPR and LTP remained impaired for at least 30 days. Treatment with the SK2 channel agonist 1-Ethyl-2-benzimidazolinone (1-EBIO) at 30 min after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post-synaptic mechanisms. Real-time quantitative reverse transcriptase-polymerase chain reaction indicated that CA/CPR did not cause a loss of N-methyl-D-aspartate (NMDA) receptor mRNA at 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels was observed at 7 or 30 days after CA/CPR. Further, patch-clamp experiments demonstrated no change in functional synaptic NMDA receptors at 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity was reduced for the duration of experiments performed (up to 30 days) and that, surprisingly, treatment with 1-EBIO did not prevent the CA/CPR-induced loss of synaptic SK channel function. We concluded that CA/CPR caused alterations in post-synaptic signaling that were prevented by treatment with the SK2 agonist 1-EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.

Keywords: cardiac arrest; global cerebral ischemia; hippocampus long-term potentiation; mouse; small conductance Ca2+-activated potassium channels.

Figure 1

Ischemia impairs synaptic plasticity in the hippocampus. A) Example fEPSPs from sham operated control and 30 day after CA/CPR mice before (black) and after (grey) TBS. B) Time course of fEPSP slope (mean ± SEM) from sham (solid circles) and mice 3 hrs (open circles) or 24 hrs (solid squares) after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Arrow indicates timing of theta burst stimulus (TBS; 40 pulses). C) Time course of fEPSP slope (mean ± SEM) from sham (solid circles) and mice 7 days (open circles) or 30 days (solid squares) after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Arrow indicates timing of theta burst stimulus (TBS; 40 pulses) D) Quantification of change in fEPSP slope following TBS. Average fEPSP slope (mean ± SEM) 60 minutes after TBS (in grey box in B normalized to baseline (black box in B). * P < 0.05 compared to sham controls.

Figure 2

CA/CPR does not affect the probability of release. A) Example traces illustrating increased amplitude and slope of fEPSP following a second stimulus administered 50 ms after the first. B) Quantification of paired-pulse ratio of sham control mice and at each time point after CA/CPR. * P < 0.05 compared to sham control. Data presented as mean ± SEM.

Figure 3

CA/CPR transiently increases slope of input-output curve (IO). A) Normalized fEPSP slope is plotted against stimulus intensity (fEPSP slope normalized to max of each recording). B) Expanded view showing increased slope at 7 days after CA/CPR compared to sham controls. No difference in IO slope was observed at 3 hrs or 30 days after CA/CPR.

Figure 4

Ischemia does not alter AMPA/NMDA ratio. A) Representative EPSCs from sham control. Application of NBQX (red trace) inhibits the AMPA component of the initial EPSC (black trace). Subsequent application of D-APV inhibits to remaining NMDA component (blue trace). B) Quantification of AMPA to NMDA ratio for all cells recorded demonstrates no difference of CA/CPR. C) Quantification of mRNA expression of the 3 predominant isoforms of NMDA receptor expressed in the hippocampus, GluN1, 2A and 2B. D) Representative Western blot analysis of NR1, PSD95 and β-actin in synaptic membrane preparations from hippocampi obtained at either 7 or 30 days after CA/CPR, or sham control mice. Quantification (bottom) of NR1:PSD95 ratio shows not changes in response to CA/CPR. Data presented as mean ± SEM.

Figure 5

Ischemia impairs SK2 channel dependent plasticity. A) Example fEPSPs from sham operated control and 30 days after CA/CPR mice in the presence of apamin before (black) and after (grey) TBS. B) Time course of fEPSP slope (mean ± SEM) from sham mice under control conditions (solid circles) and in the presence of 100 nM apamin (diamonds). Time course of fEPSP slope recorded in the presence of 100 nM apamin from mice 3 hrs (open squares) or 24 hrs (solid squares) after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Arrow indicates timing of theta burst stimulus (TBS; 40 pulses). C) Time course of fEPSP slope (mean ± SEM) from sham (solid circles) and in the presence of apamin in sham (diamond), mice 7 days (open squares) or 30 days (solid squares) after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Arrow indicates timing of theta burst stimulus (TBS; 40 pulses) CD) Quantification of change in fEPSP slope following TBS. Average fEPSP slope (mean ± SEM) 60 minutes after TBS (in grey box in B normalized to baseline (black box in B). * P < 0.05 compared to sham controls.

Figure 6

Ischemia causes sustained loss of synaptic SK channel activity in CA1 neurons. A) Quantification of mRNA expression of the SK2 channels at 24 hr, 7 and 30 days after CA/CPR. B) Time course of EPSP amplitude (mean ± SEM) from sham control mice and 7 days after CA/CPR before (black rectangle) and after (grey rectangle) a 30 minute application of 100 nM apamin. C) Quantification of the effect of apamin on EPSP amplitude. EPSP amplitude 30 minutes after application of apamin (grey square in B) normalized to baseline (black square in B). Example EPSPs from sham operated (D), 7 days after CA/CPR (E) and 30 days after CA/CPR (F) before (black) and after (red) application of apamin. *P<0.05 compared with sham controls and data presented as mean ± SEM.

Figure 7

1EBIO prevents CA/CPR loss of SK2 channel activity in synaptic plasticity. A) Time course of fEPSP slope (mean ± SEM) from sham (solid circles) and mice 30 days (open circles) after cardiac arrest and cardiopulmonary resuscitation (CA/CPR) and mice treated with 1-EBIO early after CA/CPR and time course recorded 30 days later (solid square). Arrow indicates timing of theta burst stimulus (TBS; 40 pulses). B) Time course of fEPSP slope (mean ± SEM) from 1-EBIO treated mice 30 days after CA/CPR in the presence (open square) and absence (solid square) of 100 nM apamin. Arrow indicates timing of theta burst stimuls (TBS; 40 pulses) C) Time course of fEPSP slope (mean ± SEM) from sham control mice stimulated with standard 40 pulse TBS (solid circle) or 300 pulse TBS stimulus (open square) and mice 30 days after CA/CPR stimulated with 300 pulse TBS. Arrow indicates timing of theta burst stimulus (TBS; 40 or 300 pulses) D) Quantification of change in fEPSP slope following TBS. Average fEPSP slope (mean ± SEM) 60 minutes after TBS (in grey box in A normalized to baseline (black box in A). * P < 0.05. E) Time course of EPSP amplitude (mean ± SEM) from sham control mice and 30 days after CA/CPR mice treated with 1-EBIO before (black rectangle) and after (grey rectangle) a 30 minute application of 100 nM apamin. F) Quantification of the effect of apamin on EPSP amplitude. EPSP amplitude 30 minutes after application of apamin (grey square in E) normalized to baseline (black square in E).