5-HT neurons of the area postrema become c-Fos-activated after increases in plasma sodium levels and transmit interoceptive information to the nucleus accumbens

Abstract

Serotonergic (5-hydroxytryptamine, 5-HT) neurons of the area postrema (AP) represent one neuronal phenotype implicated in the regulation of salt appetite. Tryptophan hydroxylase (Tryp-OH, synthetic enzyme-producing 5-HT) immunoreactive neurons in the AP of rats become c-Fos-activated following conditions in which plasma sodium levels are elevated; these include intraperitoneal injections of hypertonic saline and sodium repletion. Non-Tryp-OH neurons also became c-Fos-activated. Sodium depletion, which induced an increase in plasma osmolality but caused no significant change in the plasma sodium concentration, had no effect on the c-Fos activity in the AP. Epithelial sodium channels are expressed in the Tryp-OH-immunoreactive AP neurons, possibly functioning in the detection of changes in plasma sodium levels. Since little is known about the neural circuitry of these neurons, we tested whether the AP contributes to a central pathway that innervates the reward center of the brain. Stereotaxic injections of pseudorabies virus were made in the nucleus accumbens (NAc), and after 4 days, this viral tracer produced retrograde transneuronal labeling in the Tryp-OH and non-Tryp-OH AP neurons. Both sets of neurons innervate the NAc via a multisynaptic pathway. Besides sensory information regarding plasma sodium levels, the AP→NAc pathway may also transmit other types of chemosensory information, such as those related to metabolic functions, food intake, and immune system to the subcortical structures of the reward system. Because these subcortical regions ultimately project to the medial prefrontal cortex, different types of chemical signals from visceral systems may influence affective functions.

Keywords: area postrema; circumventricular organs; epithelial sodium channels; nucleus accumbens; ventral tegmental area.

Fig. 1.

Flow chart showing the design of the sodium deprivation, sodium repletion, and hypertonic saline experiments.

Fig. 2.

A: plasma sodium concentration in control rats, after 8 days of near zero-sodium diet, after 2 h of sodium repletion with either 0.9% or 3.0% NaCl following an 8-day period of zero-sodium diet, and 2 h after an intraperitoneal injection of 12% NaCl. B: bar graphs showing the plasma osmolality under the same conditions.

Fig. 3.

A: Line drawings arranged from rostral to caudal of the dorsomedial medulla from a normal rat. Note there were virtually no c-Fos-activated neurons in the area postrema (AP) or nucleus tractus solitarius (NTS). A: photoimage of the 5-HT neurons in the AP. Gr, gracile nucleus. B: 8 days of near-zero sodium diet resulted in c-Fos activation of the 11-β-hydroxysteroid dehydrogenase type 2 (HSD2) neurons in the NTS. B′: photoimage of 5-HT neurons in the AP from a rat that was sodium-deprived for 8 days. No c-Fos-activated neurons were found in the AP. C: after 8 days of near zero-sodium diet, followed by sodium repletion elicited c-Fos activation in the AP in 5-HT neurons and other AP neurons, especially in the ventrolateral AP. The neuronal phenotype(s) of the latter group is/are unknown. C′: photoimage showing 5-HT AP neurons were c-Fos activated following sodium repletion with 0.9% saline (arrows). D: sodium repletion with 3.0% saline produced a similar c-Fos activation pattern as observed in the 0.9% saline repletion experiments. Both putative 5-HT and non-5-HT neurons in the AP become c-Fos-activated. Sodium repletion elicited c-Fos activation in the medial NTS; this is likely to be the result of saline infusion into the stomach and the subsequent activation of its vagal stretch receptors. In addition, neurons in the dorsal vagal nucleus (DMX) were also c-Fos-activated, probably the result of reflex feedback from the stomach. D′: photoimage of c-Fos-activated 5-HT neurons in the AP (arrows).

Fig. 4.

A: line drawings showing the distribution of c-Fos-activated neurons in the dorsomedial medulla 2 h after an intraperitoneal injection of 12% saline. AP, area postrema; Gr, gracile nucleus; CC, central canal; DMX, dorsal vagus nucleus; t, solitary tract; NTS, nucleus tractus solitarius. B: photoimage showing 5-HT AP neurons were c-Fos-activated following an intraperitoneal injection of 12% saline. Some of the 5-HT (green) neurons were c-Fos-activated (red), as indicated by arrows.

Fig. 5.

Bar graphs showing the distribution of c-Fos-activated neurons in the AP under various sodium conditions. Note that the putative 5-HT neurons represent ∼20% of the total population of c-Fos-activated AP neurons. The data for the sodium-deprived and repletion conditions were compared with the values obtained in the control animals. In addition, data from the 12% NaCl injections are presented and compared with control values.

Fig. 6.

A: line drawing from a triple-color immunohistochemical preparation showing the distribution of neurons that expressed ENaC α-subunit (red), 5-HT (green), c-Fos (blue), and all three markers (yellow stars). This section was from a rat (case no. 6427) that was fed a near zero-sodium diet for 8 days, and then, sodium repleted for 2 h with 0.9% saline. B: photoimages showing AP neurons that expressed ENaC alpha (red), 5-HT (green), c-Fos (blue), and a merged version demonstrating all three markers were present in a single neuron.

Fig. 7.

A: photoimage of a transverse section through the rat forebrain in case no. 4545 showing an injection site in the nucleus accumbens (NAc). A cocktail of cholera toxin β-subunit (CTb) and pseudorabies virus (PRV) was injected into the core of the NAc. The CTb injection site was immediately lateral to the anterior commissure (ac). B. Four examples of injection sites in the core region of the NAc. A and B: CPu, caudate-putamen. C: Line drawings through the area postrema illustrating the distribution of 5-HT neurons (green dots), PRV-infected neurons (red dots), and colabeled neurons (yellow stars). D: photoimages showing putative 5-HT neurons, PRV-infected neurons, and colabeled neurons (merged).

Fig. 8.

A proposed central pathway showing the area postrema (AP) pathway projects by a chain of neurons to the nucleus accumbens (NAc). The AP projection is shown in red, indicating that it may provide inhibitory information to the dorsolateral pons, where it makes close contacts with the FoxP2 neurons that lie in this region (42). The two dorsolateral pontine sites include the external lateral subnucleus of the parabrachial nucleus (PB) and prelocus coeruleus nucleus (pre-LC) (28), project to the ventral tegmental area (VTA) (28). The FoxP2 neurons express mRNA for glutamate vesicular transporter, Vglut2 (Slc17a6), indicating that these neurons use glutamate as a transmitter (26), and have been shown earlier to be one of the glutamatergic afferents projecting to the ventral tegmental area (VTA) (14). This excitatory projection is shown in green. The VTA projects to the nucleus accumbens (NAc). Whether the VTA projection arises from the dopamine neurons has not yet been established. It is shown here as a double-color projection (red and green), suggesting it may produce either positive (green) or negative (red) effect on the affective system. Line drawings modified from the Paxinos rat brain atlas (34). PAG, periaqueductal gray matter; CPu, caudate-putamen; PBel-inner, external lateral subnucleus of the PB. [Adapted from Figs. 12, 38, 54, and 72 of The Rat Brain in Stereotaxic Coordinates, 2nd ed., 1986 (ISBN 9780125476218), Copyright Elsevier, Paxinos and Watson.]