Elsevier

Experimental Neurology

Volume 212, Issue 2, August 2008, Pages 415-421
Experimental Neurology

Transient cerebral ischemia increases CA1 pyramidal neuron excitability

https://doi.org/10.1016/j.expneurol.2008.04.032Get rights and content

Abstract

In human and experimental animals, the hippocampal CA1 region is one of the most vulnerable areas of the brain to ischemia. Pyramidal neurons in this region die 2–3 days after transient cerebral ischemia whereas other neurons in the same region remain intact. The mechanisms underlying the selective and delayed neuronal death are unclear. We tested the hypothesis that there is an increase in post-synaptic intrinsic excitability of CA1 pyramidal neurons after ischemia that exacerbates glutamatergic excitotoxicity. We performed whole-cell patch-clamp recordings in brain slices obtained 24 h after in vivo transient cerebral ischemia. We found that the input resistance and membrane time constant of the CA1 pyramidal neurons were significantly increased after ischemia, indicating an increase in neuronal excitability. This increase was associated with a decrease in voltage sag, suggesting a reduction of the hyperpolarization-activated non-selective cationic current (Ih). Moreover, after blocking Ih with ZD7288, the input resistance of the control neurons increased to that of the post-ischemia neurons, suggesting that a decrease in Ih contributes to increased excitability after ischemia. Finally, when lamotrigine, an enhancer of dendritic Ih, was applied immediately after ischemia, there was a significant attenuation of CA1 cell loss. These data suggest that an increase in CA1 pyramidal neuron excitability after ischemia may exacerbate cell loss. Moreover, this dendritic channelopathy may be amenable to treatment.

Introduction

Ischemic stroke is the third leading cause of death and the number one cause of disability in the United States. Selective neuronal damage in the hippocampal CA1 region occurs 2–3 days after transient cerebral ischemia (Kirino, 1982, Pulsinelli et al., 1982). A fundamental process believed to be responsible for the pathogenesis of reperfusion injury is glutamate excitotoxicity: over-stimulation of the excitatory glutamatergic receptors leads to a toxic build-up of intracellular Ca2+ that triggers cell death cascade (Urban et al., 1989, Choi and Rothman, 1990, Kirino et al., 1992, Tsubokawa et al., 1992, Hori and Carpenter, 1994, Tsubokawa et al., 1994, Xu et al., 1999). Facilitation of glutamatergic transmission is in part caused by altered biophysical and biochemical properties of the post-synaptic NMDA and AMPA receptors (Pellegrini-Giampietro et al., 1992, Crepel et al., 1993).

Another important yet poorly understood determinant of membrane depolarization upon glutamatergic synaptic stimulation is the intrinsic excitability of the CA1 pyramidal neurons. Recent advances in electrophysiological techniques have shown that dendrites are the principal sites for synaptic integration due to their non-uniform expression of a variety of voltage-gated ion channels. An interplay of these channel conductances dynamically shapes neuronal excitability hence input–output coupling (Johnston et al., 1996, Reyes, 2001). Thus, dendritic conductances could represent a novel target for studying post-ischemic excitability changes in CA1 pyramidal neurons. One prominent dendritic conductance is the hyperpolarization-activated cationic channel (h-channel) (Notomi and Shigemoto, 2004). It is composed of HCN1 and HCN2 subunits and conducts a mixed inward cationic current carried by Na+/K+ (Ih) (Robinson and Siegelbaum, 2003). Incoming excitatory post-synaptic potentials (EPSPs) can be affected by dendritic Ih through two ways: first, tonic activation of Ih enhances shunting conductance (i.e., decrease membrane input resistance), decreases membrane time constant and shortens length constant, thereby reducing the amplitudes and speeding the electrotonic decay of EPSPs (Magee, 1999); secondly, voltage-dependent deactivation of Ih upon synaptic depolarization results in a net hyperpolarizing effect that dampens the incoming EPSPs, particularly the NMDA component (Magee, 1998, Otmakhova and Lisman, 2004).

It has been suggested that Ih is an important homeostatic mechanism of regulating neuronal input–output coupling (van Welie et al., 2004, Fan et al., 2005). Dysfunction of this negative feedback mechanism has been implicated in the pathogenesis of epilepsy (Poolos, 2004, Shah et al., 2004, Zhang et al., 2006). Consistent with this, an anticonvulsant, lamotrigine, has been shown to affect neuronal excitability through up-regulating the dendritic Ih (Poolos et al., 2002). Interestingly, lamotrigine has also been shown to be neuroprotective in cerebral ischemia (Shuaib et al., 1995, Wiard et al., 1995, Crumrine et al., 1997). These findings suggest that Ih could be important for the pathogenesis of ischemic neuronal death.

In this study, we used a rodent model of transient cerebral ischemia and performed patch-clamp recordings in hippocampal slices. We report a decrease in Ih in hippocampal CA1 pyramidal neurons after transient cerebral ischemia. This, in turn, is associated with an increase in excitability. We propose that this increase in excitability may exacerbate glutamatergic excitotoxicity. Moreover, enhancing Ih may represent a novel target for the reduction of ischemic-induced neuronal loss.

Section snippets

Animal model of transient cerebral ischemia

Transient cerebral ischemia was induced in halothane-anesthetized rats by the 4-vessel occlusion protocol (Pulsinelli and Brierley, 1979, Xu and Pulsinelli, 1994). This protocol was approved by the Institutional Animal Research Committee of Baylor College of Medicine. Briefly, male adult Wistar rats (5–7 week old; Charles Rivers Laboratories) were fasted overnight to provide uniform blood glucose levels. The vertebral arteries were electrocauterized and two carotid arteries were occluded for ~ 

Increased membrane excitability after transient cerebral ischemia

To examine membrane intrinsic excitability, we performed somatic whole-cell current-clamp recordings in brain slices obtained from control animals and ischemic animals at 24 h after reperfusion. A total of 4 rats were used for the control group, and 4 rats were used for the ischemic group.

The resting membrane potentials of the ischemic neurons tended to be more depolarized than the control neurons, which is consistent with a previous report (Tsubokawa et al., 1992), However, the difference was

Discussion

This study demonstrates that there is an increase in membrane excitability of CA1 pyramidal neurons at 24 h after transient cerebral ischemia. Such an increase can be normalized to control level (i.e., non-ischemic neurons) by specific Ih blocker ZD7288, suggesting that a decrease of Ih is underlying the increase in membrane excitability. Pharmacologically enhancing Ih with LTG after the ischemia significantly attenuates cell loss. Given the predominant localization of Ih in the dendrites, our

Acknowledgments

We thank Dr. Nicholas Poolos for the critical comments and valuable suggestions on the manuscript. Y. Fan is a recipient of a post-doctoral fellowship award from the American Heart Association (0525051Y).

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