Acute stress diminishes M-current contributing to elevated activity of hypothalamic-pituitary-adrenal axis
Introduction
The hypothalamic–pituitary–adrenal (HPA) axis is critical in maintaining homeostasis as the body responds to environmental stressor (Lupien et al., 2009, McEwen, 2007, Pedersen et al., 2001). The HPA axis include the paraventricular nucleus (PVN) of the hypothalamus, which secretes corticotrophin-releasing hormone (CRH) and arginine vasopressin; the pituitary gland which releases corticotrophin (ACTH), a process triggered by CRH and arginine vasopressin; and the adrenal gland cortex, that secretes the glucocorticoids (Goncharova, 2013). As a key component of the HPA axis in basal conditions and in response to stress, the PVN-CRH neurons synthesize and release CRH, a peptide of 41 amino acid residues, and project to the median eminence (Vale et al., 1981), where CRH is released into the portal system of the pituitary (Aguilera and Liu, 2012). Acute restraint stress causes an increase in CRH mRNA levels and c-fos expression in the CRH neurons in the PVN (Day et al., 2005, Girotti et al., 2006, Imaki et al., 1998). However, the cellular mechanisms underlying the hyperactivity of the PVN-CRH neurons under stress conditions are not clear.
It has been challenging to functionally analyze CRH neuronal activity until recently, when a genetic approach to tagging CRH neuron by expressing green fluorescent protein (GFP) in transgenic mouse line was developed (Alon et al., 2009, Itoi et al., 2014, Martin et al., 2010, Wamsteeker Cusulin et al., 2013). To target CRH neurons in rat PVN, we used a recently developed approach for reliably express enhanced GFP (eGFP) driven by rat Crh promoter (Gao et al., 2017). The intrinsic neuronal excitability is tightly controlled by the transmembrane ionic currents including M-current, a voltage-gated and non-inactivating K+ current (Brown and Adams, 1980, Delmas and Brown, 2005, Marrion, 1997, Peters et al., 2005). The M-current stabilizes the membrane potential and helps maintain the resting membrane potential of neurons (Brown and Adams, 1980). Kcnq genes encode Kv7.1–7.5 K+ channel subunits, which form Kv7 channels (Brown and Yu, 2000, Brown and Adams, 1980). Genetic ablation of or acute inhibition of Kv7 channels leads to depolarization and excitation, whereas opening of Kv7-channels results in hyperpolarization and inhibition of neurons. The neuronal M-current is predominantly carried by heterotetrameric Kv7.2 and Kv7.3 subunits (Shah et al., 2002, Wang et al., 1998). Dysfunction of Kv7-channels results in several neuron-generated diseases including epilepsy, pain, memory deficit/decline, and depression (Cavaliere et al., 2013, Passmore et al., 2003, Qi et al., 2014, Zhang et al., 2013). The Kv7-channel is also involved in the regulation of a stress-related neuronal process. In this regard, activation of Kcnq/Kv7 channels prevents acute stress-induced impairments of hippocampal long-term potentiation and spatial memory retrieval in rats (Li et al., 2014).
AMP-activated protein kinase (AMPK) is a ubiquitous serine/threonine kinase which is involved in cellular responses to many metabolic stresses (Kim et al., 2009). AMPK is involved in many cellular processes, such as regulation of apoptosis, stimulation of autophagy and phagocytosis, inhibition of cell growth and proliferation, and counteraction of hypertrophy (Dermaku-Sopjani et al., 2014, Hardie, 2003). Acute restraint stress increases AMPK activity (Marques et al., 2012) and AMPK activation in the central nervous system mediates fructose-induced elevation of plasma corticosterone (CORT) levels (Kinote et al., 2012). Furthermore, AMPK activation decreases membrane expression of Kv7.1 and epithelial Na+ channel through promoting endocytosis and degradation in lysosomes via a Nedd2-4-dependent mechanism (Andersen et al., 2012, Bhalla et al., 2006). Nedd4-2 is an ubiquitin ligase that ubiquitylates membrane proteins to increase protein internalization and degradation (Abriel and Staub, 2005). Nedd4-2 suppresses Kv7.2/7.3-mediated M-currents (Ekberg et al., 2007), indicating that AMPK-Nedd4-2 is a potential pathway through which acute stress regulates Kv7 channels in PVN-CRH neuron. Thus, in this study, we tested the hypothesis that acute stress suppresses the Kv7 channels to stimulate CRH neurons through activation of AMPK.
Section snippets
Method and materials
Male Sprague-Dawley rats (12-week old) were used in this study. The rats were group-housed (n = 3 rats per cage) in a 12-h light/dark cycle and maintained under controlled temperature (24–25 °C) with food and water ad libitum. The surgical procedures and experimental protocols were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center and conformed to the National Institutes of Health guidelines on the ethical use of animals.
Acute stress blunted the excitatory effect of Kv7 channel blocker on firing activity in PVN-CRH neurons
We first identified PVN-CRH neurons expressing eGFP drive by AAV2-Crh promoter (Fig. 1A). All eGFP-tagged neurons (green) were CRH immunoreactivity positive (red) (Fig. 1B). The eGFP-tagged neurons in brain slices were viewed by using fluorescent microscope (Fig. 1C). To determine the distribution of Kv7.2 and Kv7.3 channels in the PVN-CRH neurons, we performing immunohistochemical staining with antibodies against CRH and Kv7.2 or Kv7.3. All negative controls displayed no detectable staining.
Discussion
This study determined the cellular mechanism underlying hyperactivity of PVN-CRH neuron in acute stress. We found that acute restraint stress reduced the M-current in identified PVN-CRH neurons and downregulated the expression level of Kv7.3, a subunit consisting of heterotetrameric neuronal Kv7-channels, in the PVN. Furthermore, we found that acute stress blunted Kv7-channels function in the control of circulating CORT levels and neuronal activity of PVN-CRH neurons. These data suggest that
Funding and disclosure
This study was supported by National Institute of Mental Health (NIMH) grants MH096086, and by an IRG grant from MD Anderson Cancer Center. This project was also supported by the NIH/NCI under award number P30CA016672.
Author contributions
D-P. L., Z., Z., and T.A. K. designed the study. Y-G. G, J-J. Z, Z. Z, and D-P. L. performed the experiments and analyzed the data. J-J. Z, and D-P. L wrote the manuscript. T.A. K. and Z.Z revised and commented on the manuscript.
Competing financial interests
The authors declare no competing financial interests.
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