Chronic Unpredictable Mild Stress Induces Loss of GABA Inhibition in Corticotrophin-Releasing Hormone-Expressing Neurons through NKCC1 Upregulation

Abstract

Introduction: Prolonged and repeated stresses cause hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. The corticotrophin-releasing hormone (CRH)-expressing neurons in the hypothalamic paraventricular nucleus (PVN) are an essential component of the HPA axis. Materials and Methods: Chronic unpredictable mild stress (CUMS) was induced in Sprague-Dawley rats. GABA reversal potentials (E_GABA) were determined by using gramicidin-perforated recordings in identified PVN-CRH neurons through expressing enhanced green fluorescent protein driven by the CRH promoter. Plasma corticosterone (CORT) levels were measured in rats implanted with a cannula targeting the lateral ventricles and PVN. Results: Blocking the GABA_A receptor in the PVN with gabazine significantly increased plasma CORT levels in unstressed rats but did not change CORT levels in CUMS rats. CUMS caused a depolarizing shift in E_GABA in PVN-CRH neurons compared with E_GABA in PVN-CRH neurons in unstressed rats. Furthermore, CUMS induced a long-lasting increase in expression levels of the cation chloride cotransporter Na⁺-K⁺-Cl^--Cl^- (NKCC1) in the PVN but a transient decrease in expression levels of K⁺-Cl^--Cl^- in the PVN, which returned to the basal level 5 days after CUMS treatment. The NKCC1 inhibitor bumetanide decreased the basal firing activity of PVN-CRH neurons and normalized E_GABA and the gabazine-induced excitatory effect on PVN-CRH neurons in CUMS rats. In addition, central administration of bumetanide decreased basal circulating CORT levels in CUMS rats. Conclusions: These data suggest that chronic stress impairs GABAergic inhibition, resulting in HPA axis hyperactivity through upregulation of NKCC1.

Subsequent maternal separation exacerbates neurobehavioral abnormalities in rats neonatally exposed to sevoflurane anesthesia

“…These behavioral deficiencies, taken together with the increases in hypothalamic NKCC1/KCC2 mRNA ratio and CRH mRNA levels 4 days after exposure to sevoflurane, the age period when maternal separation was imposed, suggest a role for accumulating effects of sevoflurane and maternal separation. Sevoflurane-induced up-regulation of the NKCC1/KCC2 ratio in the hypothalamus, associated with increase in GABA A R-mediated stimulation — which may further be potentiated by stressors [31,32] — may represent a mechanistic basis for the cumulative effect of neonatal anesthesia and maternal separation observed in this study. Our recent findings in rats that were neonatally exposed to a relatively short anesthesia with etomidate, another general anesthetic that enhances GABA A R activity, and several days later to a single episode of maternal separation demonstrate that such increases in the hypothalamic NKCC1/KCC2 mRNA ratio and CRH mRNA level may persist into adulthood [34].…”

Acute stress diminishes M-current contributing to elevated activity of hypothalamic-pituitary-adrenal axis

“…The bilateral guide cannula for PVN injection was affixed to the skull by using dental acrylic, a dummy cannula was inserted into each side of the guide cannula, and a dust cap was then placed over the external end of the dummy cannula. After a 1-week recovery period, the dummy cannula was removed and a bilateral injection cannula with tips protruding 1.0 mm beyond the tip of the guide cannula was inserted into the guide cannula (Gao et al, 2016). Kv7 blocker 10, 10-bis (4-pyridinylmethyl)-9(10H)-anthracnose (XE-991, 0.5 pmol in 100 nl of aCSF) was dissolved in aCSF and injected bilaterally into the PVN.…”

“…We selectively identified CRH-expressing neurons in rat PVN by expressing enhanced green fluorescent protein (eGFP) driven by rat Crh promoter (Gao et al, 2016). The full-length promoter fragment (−2125/+94) and necessary components were subcloned into the adeno-associated virus (AAV, serotype 1/2) vector expression cassette.…”

“…To enhance CRH immunoreactivity in the PVN, rats were injected intracerebroventricularly with 50–60 mg of colchicine (Gao et al, 2016; Sawchenko et al, 1984). Briefly, rats were deeply anesthetized (with sodium pentobarbital 50 mg/kg by intraperitoneal injection) and rapidly perfused transcardially with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4).…”

“…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, 2016). 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).…”