A comparison of physiological and transcriptome responses to water deprivation and salt loading in the rat supraoptic nucleus

Abstract

Salt loading (SL) and water deprivation (WD) are experimental challenges that are often used to study the osmotic circuitry of the brain. Central to this circuit is the supraoptic nucleus (SON) of the hypothalamus, which is responsible for the biosynthesis of the hormones, arginine vasopressin (AVP) and oxytocin (OXT), and their transport to terminals that reside in the posterior lobe of the pituitary. On osmotic challenge evoked by a change in blood volume or osmolality, the SON undergoes a function-related plasticity that creates an environment that allows for an appropriate hormone response. Here, we have described the impact of SL and WD compared with euhydrated (EU) controls in terms of drinking and eating behavior, body weight, and recorded physiological data including circulating hormone data and plasma and urine osmolality. We have also used microarrays to profile the transcriptome of the SON following SL and remined data from the SON that describes the transcriptome response to WD. From a list of 2,783 commonly regulated transcripts, we selected 20 genes for validation by qPCR. All of the 9 genes that have already been described as expressed or regulated in the SON by osmotic stimuli were confirmed in our models. Of the 11 novel genes, 5 were successfully validated while 6 were false discoveries.

Keywords: neuroendocrine; salt load; supraoptic nucleus; transcriptome; water restriction.

Fig. 1.

A: mean cumulative fluid intake (ml/100 g) of rats exposed to either 2% salt (SL; wt/vol) or tap water (EU) for 7 days. B: daily urine osmolality (mosmol/kg H₂O) in water-deprived (WD; 3 days) and SL animals; C: plasma osmolality in EU animals after 3 days WD and after 7 days SL (means ± SE, significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 2.

A: mean food intake in rats exposed to either 2% SL (wt/vol) for 7 days or WD for 3 days (means ± SE, significance compared with EU control). B: mean weight (g) of rats before and after either SL or WD (means ± SE, significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 3.

Plasma levels (pg/ml) of vasopressin (A), oxytocin (B), atrial natriuretic peptide (ANP, C), and angiotensin II (D) were measured from WD and SL animals (one-way ANOVA, *P < 0.05, **P < 0.005, ***P < 0.0001).

Fig. 4.

A: mean cumulative fluid intake (ml/100 g) of rats replete for water following an osmotic challenge of either 2% salt (wt/vol; SL) for 7 days or WD for 3 days compared with EU animals (means ± SE) B: mean weight (g) of SL, WD, and EU animals after osmotic challenge and after repletion (means ± SE). Significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.

Affymetrix microarrays were used to interrogate supraoptic nucleus (SON) from EU vs. 7-day SL animals (2% wt/vol) and revealed 9,843 genes (A) significantly regulated genes (Welch t-test, corrected using Benjamini-Hochberg to keep P < 0.05, S1), of which 1,108 genes (B) were regulated by greater than twofold (S2) between EU and SL. Microarrays comparing EU and 3-day WD in the SON were also compared, and 4,480 genes (S3) were regulated of which 106 genes (S4) were regulated by greater than twofold. In total, 2,783 genes (S5) were commonly regulated by both SL and WD in the rat SON, and 95 genes (S6) that were commonly regulated by greater than 2-fold (see supplementary Figs. S1–S6).

Fig. 6.

Probes identified by the array and validation by PCR in the SON. FC, Fold change, NS, not significant, Ref, references to support gene expression in the SON. Significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7.

Quatitative PCR (qPCR) validation of mRNA expression (ΔΔC_t) in the SON of the EU, 7-day SL, and the 3-day WD rat (means ± SE, significance = paired t-test, *P < 0.05). See text for specific details about A–T. Trvp, transient receptor potential cation channel V2; Arhgdib; Rho, GDP dissociation inhibitor (GDI) β; Atf4, activating transcription factor 4; Creb3l1, cAMP-responsive element binding protein 3-like 1; Giot1, gonadotrophin-inducible ovarian transcription factor 1; Opn3, Opsin3; Vgf, VGF nerve growth factor inducible; Syt4, synaptotagmin IV; Rgs4, regulator of G protein signaling 4; Cd55, Cd55 molecule/complement decay-accelerating factor; Nab1, nerve growth factor inducible-A-binding protein 1; Procr, endothelial protein C receptor; Atf5, activating transcription factor 5; Psph, phosphoserine phosphatase; Itgb1, integrin, β1; Rgs2, regulator of G protein signaling 2; Insig1, insulin-induced gene 1; Rgs5, regulator of G protein signaling 5. Significance: NS, not significant *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.