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. 2009 Sep;219(1):212-22.
doi: 10.1016/j.expneurol.2009.05.019. Epub 2009 May 20.

Neurotransmitter regulation of c-fos and vasopressin gene expression in the rat supraoptic nucleus

Makoto Kawasaki  1 , Todd A Ponzio, Chunmei Yue, Raymond L Fields, Harold Gainer
Affiliations

Neurotransmitter regulation of c-fos and vasopressin gene expression in the rat supraoptic nucleus

Makoto Kawasaki et al. Exp Neurol. 2009 Sep.

Abstract

Acute increases in plasma osmotic pressure produced by intraperitoneal injection of hypertonic NaCl are sensed by osmoreceptors in the brain, which excite the magnocellular neurons (MCNs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) in the hypothalamus inducing the secretion of vasopressin (VP) into the general circulation. Such systemic osmotic stimulation also causes rapid and transient increases in the gene expression of c-fos and VP in the MCNs. In this study we evaluated potential signals that might be responsible for initiating these gene expression changes during acute hyperosmotic stimulation. We use an in vivo paradigm in which we stereotaxically deliver putative agonists and antagonists over the SON unilaterally, and use the contralateral SON in the same rat, exposed only to vehicle solutions, as the control SON. Quantitative real time-PCR was used to compare the levels of c-fos mRNA, and VP mRNA and VP heteronuclear (hn)RNA in the SON. We found that the ionotropic glutamate agonists (NMDA plus AMPA) caused an approximately 6-fold increase of c-fos gene expression in the SON, and some, but not all, G-coupled protein receptor agonists (e.g., phenylephrine, senktide, a NK-3-receptor agonist, and alpha-MSH) increased the c-fos gene expression in the SON from between 1.5 to 2-fold of the control SONs. However, none of these agonists were effective in increasing VP hnRNA as is seen with acute salt-loading. This indicates that the stimulus-transcription coupling mechanisms that underlie the c-fos and VP transcription increases during acute osmotic stimulation differ significantly from one another.

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Figures

Figure 1
Figure 1
Convection enhanced delivery (CED) and ALZET osmotic minipump methods were used to stereotaxically deliver various pharmacological agents to the supraoptic nucleus (SON) in vivo. (A) Illustrates a schematic diagram of this paradigm showing in a rat brain coronal section at the level of mid SON. The locations of the bilateral cannulae positioned approximately 300 μm over each SON. (B) Magnocellular neurons (MCNs) in the SON are shown by immunofluorescence in red using the PS45 antibody that cross-reacts with both vasopressin (VP) and oxytocin (OT)-NP. Fluoro-Gold (0.005%) was delivered by CED to one SON (right panel) and 0.9% saline was delivered to the contralateral SON (left panel). The Fluoro-Gold filling the SON and surrounding area is shown as blue fluorescence, and the dotted line shows the full extent of the dye spread. (C) MCNs in the SON are shown by single-label immunofluorescence in red using the PS45 antibody against VP and OT-NP. Fluoro-Gold (0.005%) was delivered by the ALZET minipump method to one SON and 0.9% saline was delivered to the contralateral SON (left panel). The extent of Fluoro-Gold filling the SON and surrounding area is shown as blue fluorescence, and the dotted line shows the extent of the dye spread. OX, optic chiasm; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus. Scale bars = 100 μm
Figure 2
Figure 2
Quantitative real time PCR (qRT-PCR) of c-fos mRNA, VP mRNA and VP heteronuclear (hn)RNA. (A) Locations of qRT-PCR primers in the RNAs are shown as arrows representing the positions of forward and reverse PCR primer binding sites (See Table 1 for sequences). In this single step assay, the reverse PCR primer also serves as the primer for reverse transcription (see Methods). (B) Representative fluorescence “growth curves” produced by quantitative real time (qRT)-PCR for c-fos mRNA, VP mRNA and VP hnRNA are shown for the SON 30 min after 0.15M NaCl intraperitoneal (i.p.) injection (control conditions).
Figure 3
Figure 3
Changes in Ct values for c-fos mRNA, VP mRNA and VP hnRNA in rat SONs after acute osmotic stimulation as measured by the qRT-PCR assay. (A) Average ± SEM Cycle threshold (Ct) valuesfrom control SONs in rats injected with 0.15M NaCl (black bars) versus SONs in rats injected with 2M NaCl (stripped bars) are shown. (B) Calculatedpercent changes in c-fos mRNA and VP hnRNA in the SON 30 min after the injection of hyperosmotic 2M compared to rats injected with isotonic 0.15M NaCl injection. Statistically significant differences between Ct values in the 0.15M and 2M injected rats are shown by asterisks where P< 0.05.
Figure 4
Figure 4
Inhibition of voltage-sensitive sodium channels in SON by TTX delivered by ALZET osmotic minipump blocks increased expression of c-fos mRNA and VP hnRNA in response to acute hyperosmotic stimulation by 2M NaCl i.p. injection. (A) The decreases in c-fos mRNA and VP hnRNA in the TTX-treated (3.5h) SON after acute osmotic stimulus (30 min) compared to their increases the control SON are illustrated as a percent of control SON values. The reductions of c-fos mRNA and VP hnRNA in the TTX-treated SON were 64% and 41% of the control SON, respectively. (B) Double immunostaining for Fos (in Red) and for both VP and OT-MCNs (PS45-ir, Green) after i.p. injection of hypertonic NaCl in rats. The PBS (Control) side (B-I) expressed Fos in the SON, but the TTX-treated side (B-II) did not express Fos, confirming the inhibition of Fos expression by ALZET osmotic minipump delivery of TTX. Scale bars=50 μM
Figure 5
Figure 5
Effects of direct depolarization and hyperosmotic stimulation on c-fos mRNA and VP hnRNA in the SON. (A) Veratridine (100 μM) applied directly to the SON by CED increased the expression of c-fos fourfold but did not increase VP transcription in the SON. (B) Direct osmotic stimulation of the MCNs in SON by direct application of 494 mOsm/kg hyperosmotic mannitol to the SON did not significantly change the expression of either the c-fos or the VP gene in the SON.
Figure 6
Figure 6
Direct application of an excitatory cocktail (NAB) consisting of ionotropic-glutamate receptor agonists (NAB), NMDA (100 μM), AMPA (100 μM) plus bicuculline (500 μM), a GABAA receptor antagonist to the SON by CED increased the expression of c-fos sixfold, but did not change VP gene expression in the SON.
Figure 7
Figure 7
Ionotropic glutamate antagonists do not significantly block either c-fos mRNA or VP hnRNA expression in response to systemic hyperosmotic stimulation. The osmotically induced increases in c-fos and VP gene expression by i.p. injection of 2M NaCl (see Fig. 3) is not significantly inhibited by the direct application of glutamate antagonists, CNQX (>100 μM) and APV (>100 μM) by ALZET osmotic minipump delivery to the SON.
Figure 8
Figure 8
Effects of the direct application of various G-protein coupled receptor (GPCR) agonists by CED on c-fos and VP gene expression in the SON. (A) Phenylephrine (100 μM) plus ATP (1 mM), (B) Senktide (Substance P analogue) (100 μM) plus ATP (1 mM), and (C) alpha-melanocyte stimulating hormone (α-MSH) (500 μM) all increased the expression of c-fos mRNA but not VP hnRNA in the SON. (D) Angiotensin II did not increase the gene expression of either c-fos or VP.
Figure 9
Figure 9
Test of possible synergistic effects by the combined actions of excitatory amino acids and selected GPCR agonists on c-fos and VP gene expression in the SON. Agonist cocktail containing NAB (NMDA (100 μM), AMPA (100 μM), bicuculline (500 μM)), phenylephrine (100 μM), senktide (100 μM), α-MSH (410 μM) and ATP (1 mM) was delivered by CED increased the expression of c-fos but not the VP gene in the SON.
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