Dilutional hyponatremia due to increased plasma arginine vasopressin (AVP) is associated with liver cirrhosis. However, plasma AVP remains elevated despite progressive hypoosmolality. This study investigated changes to inhibitory control of supraoptic nucleus (SON) AVP neurons during liver cirrhosis. Experiments were conducted with adult male Sprague-Dawley rats. Bile duct ligation was used as a model of chronic liver cirrhosis. An adeno-associated virus containing a construct with an AVP promoter and either green fluorescent protein (GFP) or a ratiometric chloride indicator, ClopHensorN, was bilaterally injected into the SON of rats.After 2 weeks, rats received either BDL or sham surgery, and liver cirrhosis was allowed to develop for 4 weeks. In vitro, loose patch recordings of action potentials were obtained from GFP-labeled and unlabeled SON neurons in response to a brief focal application of the GABA A agonist muscimol (100 μM). Changes to intracellular chloride ([Cl]i) following muscimol application were determined by changes to the fluorescence ratio of ClopHensorN. The contribution of cation chloride cotransporters NKCC1 and KCC2 to changes in intracellular chloride was investigated using their respective antagonists, bumetanide (BU, 10 μM) and VU0240551 (10 μM). Plasma osmolality and hematocrit were measured as a marker of dilutional hyponatremia. The results showed reduced or absent GABA A -mediated inhibition in a greater proportion of AVP neurons from BDL rats as compared to sham rats (100% inhibition in sham vs. 47% in BDL, p = .001).Muscimol application was associated with increased [Cl]i in most cells from BDL as compared to cells from sham rats (χ 2 = 30.24, p < .001). NKCC1 contributed to the impaired inhibition observed in BDL rats (p < .001 BDL À BU vs. BDL + BU).The results show that impaired inhibition of SON AVP neurons and increased intracellular chloride contribute to the sustained dilutional hyponatremia in liver cirrhosis.
Dilutional hyponatremia typically is caused by increased Arginine Vasopressin (AVP) secretion. This increases the risk of complications and mortality in patients with liver cirrhosis. Liver cirrhosis in male rats increases AVP release but the underlying mechanisms are still under investigation. In other models of chronic AVP release, changes in chloride homeostasis alters the inhibitory control of AVP neurons in the supraoptic nucleus (SON) causing GABA mediated excitation instead of inhibition. In the bile duct ligation (BDL) model of cirrhosis, changes in either intracellular chloride concentration or cell firing activity have not been directly tested. We hypothesize that liver cirrhosis increases intracellular chloride concentration leading to a decrease in GABA‐mediated inhibition of SON neurons from male rats. To test this hypothesis, we injected an adeno‐associated virus with a vasopressin promoter and a ratiometric chloride indicator, AAV2‐0VP1‐ClopHensorN into the SON of adult male Sprague‐Dawley rats. The rats either received BDL surgery, which was used to model liver cirrhosis, or a sham ligation surgery. All BDL rats had significantly higher liver weight to body weight ratio (n = 10‐20, p < 0.05), lower plasma osmolality (n = 10‐20, p < 0.001) and lower hematocrit (n = 10‐20, p < 0.05) as compared to sham rats. All rats were anesthetized with isoflurane (2‐3%), their brains were collected, and slices containing the SON were prepared. Loose‐cell patch recordings were made at 31±1oC using standard artificial cerebrospinal fluid. Labeled AVP cells and unlabeled SON cells were tested with focal application of muscimol (100µM), a GABAA receptor agonist. Cells from BDL rats showed impaired GABAA‐mediated inhibition in response to muscimol, as compared to those from sham rats. All putative AVP SON neurons from sham rats (4/4) showed decreased activity following muscimol while half of the putative AVP SON neurons (7/14) from BDL rats were excited (p < 0.001). Similar results were observed in experiments were the injections missed the SON. In these experiments, all SON neurons from sham rats were inhibited by muscimol (26/26) while thirty‐five percent of SON neurons from BDL rats (16/45) were excited by muscimol (p = 0.001). ClopHensorN based chloride imaging showed that muscimol application was associated with decreased intracellular chloride concentrations in putative AVP cells from BDL rats as compared to sham rats (n = 31‐32, p = 0.001). The results show that liver cirrhosis impairs the normal inhibitory response of SON neurons to GABAA receptor activation which could contribute to increased cell activity and dilutional hyponatremia in male rats.
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