Hypoxia enhances high-voltage-activated calcium currents in rat primary cortical neurons via calcineurin
Introduction
Brain neurons can tolerate moderate to severe hypoxia for several hours (Erecinska and Silver, 2001, Scheufler et al., 2002) as long as glucose is present to meet minimal metabolic demands via glycolysis (Kahlert and Reiser, 2000, Vannucci and Vannucci, 2000, Vannucci and Vannucci, 2001). However, hypoxia can predispose to seizures, which occur in 9% of patients after stroke (Bladin et al., 2000) and up to 36% of patients after cardiopulmonary arrest (Krumholz et al., 1988). The risk of epilepsy after perinatal hypoxia is 5-fold greater than in normal neonates (Bergamasco et al., 1984, Jensen, 2002). Thus, understanding the cellular mechanisms mediating hypoxia-induced neuronal dysfunction is clinically relevant and could lead to new treatments for hypoxia-induced seizures and epilepsy.
Transmembrane ion channels play important roles in oxygen (O2)-regulated cellular processes (Hammarstrom and Gage, 1998, Jiang et al., 1994, Tai and Truong, 2007). We previously reported that transient hypoxia reduces inhibitory GABAA receptor (GABAAR) function in NT2-N neurons (Gao et al., 2004) and primary cortical neurons (Wang and Greenfield, 2009) and that the reduction in cortical neurons was blocked by the L-type voltage-gated calcium channel (L-VGCC) antagonist, nitrendipine (Wang and Greenfield, 2009). L-VGCCs play a critical role in mediating intracellular Ca2+ signaling, activity-dependent synaptic plasticity and neuronal survival (Berridge, 1998, Catterall, 2000), and are likely involved in post-hypoxic signaling mechanisms. However, the sensitivity of neurons to hypoxia varies by neuron type and brain region, and cortical neurons are more resistant to O2 deprivation than hippocampal or cerebellar neurons (Krnjevic, 2008). For example, Li et al. (2007) reported a delayed reduction in Ca2+ currents in hippocampal CA1 but not CA3 neurons, associated with increased cell death in CA1 neurons. Moreover, regulation of L-VGCCs after deprivation of both O2 and glucose may differ markedly from hypoxia alone. The effects of short term hypoxia on high voltage activated (HVA) Ca2+ currents in primary rat cortical neurons have not been thoroughly investigated. Regulation of cortical neuron HVA Ca2+ currents by hypoxia may contribute to cortical hyperexcitability and thus predispose to seizures.
At least four types of HVA VGCCs (L-, N-, P/Q- and R-type) have been identified in neurons, with diverse regulatory mechanisms that are specific to VGCC subtypes (Catterall, 2000). One important regulatory factor is the Ca2+/calmodulin-dependent phosphatase, calcineurin (also known as protein phosphatase 2B). Both positive and negative regulations of VGCC activity by calcineurin have been reported, varying across cell types (Groth et al., 2003) and model systems (Zeilhofer et al., 2000, Norris et al., 2002, Oliveria et al., 2007, Norris et al., 2010), suggesting that the physiological regulation of VGCCs by calcineurin is complex and variable depending on the neurons involved. Since seizure generation is a predominantly cortical activity, it is critical to understand how seizure-inducing stimuli like hypoxia affect cortical neuron VGCCs and the possible role of calcineurin in this response.
Here, we evaluated HVA Ca2+ currents in rat primary cortical neurons in culture using whole-cell voltage clamp recordings after exposure to 1% O2 for 4 h. HVA Ca2+ currents were increased immediately (0–2 h) after 4 h hypoxia but returned to baseline when recorded after 48 h normoxic recovery. The increase in HVA current was blocked by nimodipine (NIM), and hence L-VGCC-dependent. Inhibition of calcineurin activity with FK-506 or cyclosporine A (CsA) blocked the post-hypoxic increase in L-VGCC current. Our results suggest that O2 deprivation transiently increases L-VGCC activity in cortical neurons via a calcium dependent process requiring L-VGCC activation and calcineurin, suggesting a positive feedback loop to amplify neuronal calcium signaling after hypoxia. These findings may have clinical significance, since hypoxia-induced increases in intracellular Ca2+ after stroke or cardiopulmonary arrest may contribute to post-hypoxic neuronal hyperexcitability, cell death or epileptogenesis.
Section snippets
Ethical approval
Experimental protocols involving the use of vertebrate animals were approved by the University of Toledo College of Medicine Institutional Animal Care and Use Committee (IACUC) and conformed to United States National Institutes of Health guidelines.
Cell cultures
Primary cultures of cortical neurons were prepared from E18 fetal Sprague-Dawley rats according to a protocol slightly modified from established techniques (Porter et al., 1997). Briefly, E18 rat fetuses were removed under sterile conditions after
Pharmacological characterization of HVA Ca2+ currents in rat cortical neurons
Cultured rat primary cortical neurons were inspected by phase-contrast microscopy prior to recordings to confirm normal morphology and viability after 4 h exposure to 1% O2. Consistent with our previous reports (Gao et al., 2004, Wang and Greenfield, 2009), this hypoxic exposure did not result in obvious somatic swelling or dendrite retraction, features commonly associated with neuronal injury, and showed no increase in cell death or injury as measured by trypan blue staining or LDH release (Gao
Discussion
In cultured rat cortical neurons, exposure to 1% O2 for 4 h resulted in Ca2+ entry through L-type VGCCs, activation of calcineurin and a transient increase in L-type VGCC currents that returned to baseline within 48 h after exposure. Blockade of L-type channels during hypoxia prevented the increase in HVA Ca2+ currents, suggesting a critical role for L-VGCCs in their own potentiation. Inhibition of calcineurin either by CsA during hypoxia or by FK-506 in the recording pipette after hypoxia
Grants
This work was supported in part by R01-NS049389 and a research grant from the Myoclonus Research Foundation, Ft. Lee, N.J. to L.J.G.
Conflict of interest
None.
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Current address: Department of Internal Medicine, University of Toledo College of Medicine, Toledo, OH 43614, USA.