Helmut B. Gottlieb

Differential effects of water deprivation and rehydration on Fos and FosB/ΔFosB staining in the rat brainstem

This study examined the effects of dehydration and rehydration with water on Fos and FosB staining in the brainstem of rats. Male rats were water deprived for 48 h (Dehyd, n=7) or 46 h followed by 2 h access to water (Rehyd, n=7). Controls had ad libitum access to water (Con, n=9). Brainstems were stained for Fos and FosB/DeltaFosB using commercially available antibodies. In the nucleus of the solitary tract (NTS), the number of Fos stained neurons was significantly increased by dehydration and increased further following rehydration (Con 5+/-1; Dehyd 22+/-1; Rehyd 48+/-5). The average number of Fos-positive cells in the parabrachial nucleus (PBN) was significantly increased only by rehydration (Con 12+/-2; Dehyd 6+/-2; Rehyd 51+/-4). The area postrema (AP) showed significant increases in Fos staining after dehydration and rehydration (Fos: Con 4+/-1; Dehyd 28+/-3; Rehyd 24+/-3). In the rostral ventrolateral medulla (RVL), Fos staining significantly increased after dehydration and this effect was reduced by rehydration (Con 3+/-1; Dehyd 21+/-2; Rehyd 12+/-1). In contrast, Fos staining in the caudal ventrolateral medulla (CVL) was not significantly influenced following either dehydration or rehydration with water (Con 4+/-1; Dehyd 4+/-1; Rehyd 5+/-1). FosB/DeltaFosB staining in the NTS, AP, and RVL was comparably increased by dehydration and rehydration. In the PBN and CVL, FosB/DeltaFosB staining was not affected by the treatments. Dehydration and rehydration have regionally specific effects on Fos and FosB/DeltaFosB staining in the brainstem.

ΔFosB in the supraoptic nucleus contributes to hyponatremia in rats with cirrhosis

Bile duct ligation (BDL), a model of hepatic cirrhosis, is associated with dilutional hyponatremia and inappropriate vasopressin release. ΔFosB staining was significantly increased in vasopressin and oxytocin magnocellular neurosecretory cells in the supraoptic nucleus (SON) of BDL rats. We tested the role of SON ΔFosB in fluid retention following BDL by injecting the SON (n = 10) with 400 nl of an adeno-associated virus (AAV) vector expressing ΔJunD (a dominant negative construct for ΔFosB) plus green fluorescent protein (GFP) (AAV-GFP-ΔJunD). Controls were either noninjected or injected with an AAV vector expressing only GFP. Three weeks after BDL or sham ligation surgery, rats were individually housed in metabolism cages for 1 wk. Average daily water intake was significantly elevated in all BDL rats compared with sham ligated controls. Average daily urine output was significantly greater in AAV-GFP-ΔJunD-treated BDL rats compared with all other groups. Daily average urine sodium concentration was significantly lower in AAV-GFP-ΔJunD-treated BDL rats than the other groups, although average daily sodium excretion was not different among the groups. SON expression of ΔJunD produced a diuresis in BDL rats that may be related to decreased circulating levels of vasopressin or oxytocin. These findings support the view that ΔFosB expression in SON magnocellular secretory cells contribute to dilutional hyponatremia in BDL rats.

Role of superior laryngeal nerve and Fos staining following dehydration and rehydration in the rat

Immunohistochemistry for Fos was used to determine the role of the superior laryngeal nerve in conscious rats following water deprivation and rehydration. Adult male rats were subjected to either unilateral superior laryngeal nerve section (SLNX) or sham surgery. Two weeks later rats from each surgical group were water deprived for 48 h or water deprived for 46 h and given access to water for 2 h prior to perfusion. Controls were allowed ad libitum access to water. Brains were processed for Fos using a commercially available antibody. Changes in plasma osmolality and hematocrit were not significantly different between SLNX and sham following any of the treatments. Water intake in rats was not significantly affected by SLNX. In the supraoptic nucleus (SON) of sham rats, water deprivation significantly increased Fos staining while water intake following dehydration prevented this increase. Water deprivation significantly increased Fos staining in the SON of SLNX rats. Following water intake after 46 h water deprivation in SLNX rats, Fos staining in the ipsilateral SON was significantly greater than the contralateral SON and significantly lower than 48 h water deprivation. In the nucleus of the solitary tract (NTS) of sham rats, both water deprivation and water intake produced significant increases in Fos staining bilaterally compared to euhydrated controls. In SLNX rats, water deprivation significantly increased Fos in both ipsilateral and contralateral NTS that was not different from sham rats. SLNX significantly decreased Fos staining in the ipsilateral NTS of rats given access to water after dehydration compared to the corresponding sham treated rats. Fos staining was not affected in the contralateral NTS of SLNX rats given access to water after dehydration. This suggests that the superior laryngeal nerve contributes to changes in Fos staining in the NTS and SON following water intake in dehydrated rats.

Identification of Central Nervous System Sites Involved in the Water Diuresis Response Elicited By Central Microinjection of Nociceptin/ Orphanin FQ in Conscious Rats Via c-Fos and Inducible cAMP Early Repressor Immunocytochemistry

Intracerebroventricular (i.c.v.) administration of the opioid‐like peptide, nociceptin/Orphanin (nociceptin), in conscious rats produces diuretic and antinatriuretic effects. The present study utilised changes in Fos and inducible cAMP early repressor (ICER) immunocytochemistry expression to examine the central nervous (CNS) sites activated or inhibited, respectively, by central administration of nociceptin. Urine samples were collected during control (15 min) and after i.c.v. vehicle (5 µl, n = 12) or nociceptin (10 µg/5 µl; n = 12). Four additional urine samples (15‐min) were collected after the i.c.v. injection. The brain was processed for Fos using a commercially available antibody (Oncogene AB‐5) and for ICER using a polyclonal anti‐ICER antibody raised in rabbits. In vehicle‐injected conscious rats, renal excretion of water or sodium was not altered. However, nociceptin produced a rapid and marked increase in urine flow (V) and a decrease in urinary sodium excretion rate. In addition, i.c.v. nociceptin produced a significant increase in Fos staining in the dorsomedial nucleus of the hypothalamus, the perinuclear zone of the supraoptic nucleus, the organum vasculosum of the lamina terminalis (OVLT), the lateral preoptic area and the lateral hypothalamic area compared to control. By contrast, Fos expression decreased in the area postrema and locus coeruleus compared to controls. Furthermore, ICER staining was significantly increased in the perinuclear zone of the supraoptic nucleus, supraoptic nucleus, median preoptic nucleus, OVLT, medial preoptic area, central nucleus of the amygdala, and medial nucleus of the solitary tract. Together, central opioid receptor‐like type 1 activation in these CNS regions may participate in the neural pathways involved in the diuretic and antinatriuretic effects of nociceptin.