Increased expression of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels in reactive astrocytes following ischemia

Abstract

Astrocytes respond to ischemic brain injury by proliferation, the increased expression of intermediate filaments and hypertrophy, which results in glial scar formation. In addition, they alter the expression of ion channels, receptors and transporters that maintain ionic/neurotransmitter homeostasis. Here, we aimed to demonstrate the expression of Hcn1-4 genes encoding hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following focal cerebral ischemia (FCI) or global cerebral ischemia (GCI) and to characterize their functional properties. A permanent occlusion of the middle cerebral artery (MCAo) was employed to induce FCI in adult GFAP/EGFP mice, while GCI was induced by transient bilateral common carotid artery occlusion combined with hypoxia in adult rats. Using FACS, we isolated astrocytes from non-injured or ischemic brains and performed gene expression profiling using single-cell RT-qPCR. We showed that 2 weeks after ischemia reactive astrocytes express high levels of Hcn1-4 transcripts, while immunohistochemical analyses confirmed the presence of HCN1-3 channels in reactive astrocytes 5 weeks after ischemia. Electrophysiological recordings revealed that post-ischemic astrocytes are significantly depolarized, and compared with astrocytes from non-injured brains, they display large hyperpolarization-activated inward currents, the density of which increased 2-3-fold in response to ischemia. Their activation was facilitated by cAMP and their amplitudes were decreased by ZD7288 or low extracellular Na(+) concentration, suggesting that they may belong to the family of HCN channels. Collectively, our results demonstrate that regardless of the type of ischemic injury, reactive astrocytes express HCN channels, which could therefore be an important therapeutic target in poststroke therapy.

Optimized Model of Cerebral Ischemia In situ for the Long-Lasting Assessment of Hippocampal Cell Death

“…After years of investigating neuronal vulnerability to ischemic damage through different models of mimicking ischemic insults in situ , it has been emerged that the degree of neuronal damage and ultimate cell death strictly depend on severity of such modeling (e.g., a duration of lesion). Our results of a sharp neuronal loss and overwhelmed astrogliosis within destructive CA1 area by prolonged OGD (30 min duration) also argue for that, being fully consistent with previous reports of a massive cell death by OGD lasting up to an hour (Graulich et al, 2002; Rytter et al, 2003; Jung et al, 2004; Le et al, 2015) and astrogliosis (Cho et al, 2013; Honsa et al, 2014). In contrast to 30-min OGD, mild OGD (10-min duration) produced a little destruction to the hippocampal tissue, justified by a steady level of both NeuN-positive and PI-positive cells shortly after induction of mild OGD, similar to other studies (Matsuzaki et al, 1997; Lecoeur, 2002).…”

Subanesthetic doses of ketamine stabilize the fusion pore in a narrow flickering state in astrocytes

“…; Honsa et al . ) and changes in local cation concentrations mediated by HCN channels, activated by cAMP, play a role in modulating the fusion pore (Calejo et al . ).…”

“…Although ketamine is commonly known as an NMDAR antagonist, ketamine also targets hyperpolarization-activated cyclic nucleotide-gated (HCN1) channels that are implicated in its hypnotic actions (Chen et al 2009) and as therapeutic targets for depressive disorders (Shah 2012). Astrocytes express HCN1 channels (Rusnakova et al 2013;Honsa et al 2014) and changes in local cation concentrations mediated by HCN channels, activated by cAMP, play a role in modulating the fusion pore (Calejo et al 2014). Also cAMP has been shown to stabilize an intermediate stage where the vesicle fusion pore transiently opens, thus increasing the probability of its rhythmic reopening (Calejo et al 2013).…”

HCN2 ion channels: basic science opens up possibilities for therapeutic intervention in neuropathic pain

“…In contrast, the expression of HCN3 and HCN4 in the CNS appears less abundant. Interestingly, I h currents have also been reported in reactive brain astrocytes as well as pain-processing neurons in the spinal cord [90][91][92]. Interestingly, I h currents have also been reported in reactive brain astrocytes as well as pain-processing neurons in the spinal cord [90][91][92].…”