Area postrema projects to FoxP2 neurons of the pre-locus coeruleus and parabrachial nuclei: brainstem sites implicated in sodium appetite regulation

Abstract

The area postrema (AP) is a circumventricular organ located in the dorsal midline of the medulla. It functions as a chemosensor for blood-borne peptides and solutes, and converts this information into neural signals that are transmitted to the nucleus tractus solitarius (NTS) and parabrachial nucleus (PB). One of its NTS targets in the rat is the aldosterone-sensitive neurons which contain the enzyme 11 β-hydroxysteroid dehydrogenase type 2 (HSD2). The HSD2 neurons are part of a central network involved in sodium appetite regulation, and they innervate numerous brain sites including the pre-locus coeruleus (pre-LC) and PB external lateral-inner (PBel-inner) cell groups of the dorsolateral pons. Both pontine cell groups express the transcription factor FoxP2 and become c-Fos activated following sodium depletion. Because the AP is a component in this network, we wanted to determine whether it also projects to the same sites as the HSD2 neurons. By using a combination of anterograde axonal and retrograde cell body tract-tracing techniques in individual rats, we show that the AP projects to FoxP2 immunoreactive neurons in the pre-LC and PBel-inner. Thus, the AP sends a direct projection to both the first-order medullary (HSD2 neurons of the NTS) and the second-order dorsolateral pontine neurons (pre-LC and PB-el inner neurons). All three sites transmit information related to systemic sodium depletion to forebrain sites and are part of the central neural circuitry that regulates the complex behavior of sodium appetite.

Figure 1

Transverse brainstem section showing the distribution of FoxP2-ir neurons in the parabrachial nucleus (PB). A. The insert shown in the upper left hand corner presents an enlargement of the FoxP2-ir neurons in the external lateral parabrachial subnucleus- inner division (PBel-inner). Other abbreviations: KF= Kölliker-Fuse nucleus; PBdl= dorsal lateral parabrachial subnucleus; PBm= medial parabrachial subnucleus; MeV = mesencephalic nucleus of the trigeminal nerve; mtV = tract of the mesencephalic trigeminal nucleus; scp= superior cerebellar peduncle. B. Transverse brainstem section showing the pre-locus coeruleus nucleus (pre-LC). This nucleus lies immediately rostral to the locus coeruleus within the lateral part of the periventricular gray matter. As shown in the enlargement in the upper left hand corner, the FoxP2-ir neurons are concentrated in the zone medial to the mesencephalic nucleus of the trigeminal nerve (MeV), but some are interspersed among the MeV neurons..

Figure 2

A–D: PHAL injection sites in the area postrema along with accompanying line drawings. All cases presented here had injections confined to the area postrema without any cell body labeling in the subpostremal or medial NTS. Drawings were modified from Paxinos and Watson (2005). Abbreviations: AP = area postrema; CC = central canal; DMX = dorsal vagal nucleus; Gr = gracile nucleus; NTS = nucleus tractus solitarius; t = solitary tract; SubP = subpostremal NTS region.

Figure 3

A–D. Photoimages and accompanying line drawings showing the distribution of PHAL labeled axons (red) among the FoxP2-ir neurons in the parabrachial nucleus (PB) and pre-locus coeruleus nucleus (pre-LC) after a PHAL injection in the area postrema. Sections are arranged from rostral (A) to caudal (D). Panel A shows additional axonal labeling that was medial to the mesencephalic nucleus of the trigeminal nerve (MeV); this area was independent of the pre-LC. Panel B shows very weak axonal labeling in the PB external lateral subnucleus. Panel C shows a dense cluster of labeled axons in the PB external lateral-inner division (PB-el inner). An occasional labeled axon was found in other parabrachial subnuclei including the PB central lateral (PBcl), PB dorsal lateral subnucleus (PBdl), and outer division of the PB external lateral subnucleus (C and D). Panel D shows a dense collection of labeled axons surrounding the FoxP2-ir neurons of the pre-LC. Additional abbreviations: mtV= tract of the mesencephalic nucleus of the trigeminal nerve; PBm = medial PB subnucleus; scp = superior cerebellar peduncle. Scale bar = 100 μm.

Figure 4

The experimental design of the double tract-tracing experiments used to examine whether the AP projection neurons make close contacts with the FoxP2-ir neurons in the pre-LC. The anterograde axonal tracer PHAL (red) was injected into the area postrema, and 3 days later, the retrograde cell body marker CTb (blue) was injected into one of several forebrain sites, including the bed nucleus of the stria terminalis (as shown here) or the dorsomedial, paraventricular, and perifornical areas of the hypothalamus. Sections through the dorsolateral pons were processed by a triple immunofluorescence method and examined by confocal microscopy.

Figure 5

Double tract-tracing experiment to demonstrate that AP neurons project to FoxP2-ir neurons of the pre-LC that send efferent connections to the bed nucleus of the stria terminalis (BST). A–B. A CTb injection (blue) was made in the BST andPHAL injection (red) into the area postrema (AP). C. 20× confocal image of the pre-LC region showing PHAL axons in relationship to FoxP2-ir (green) and CTb labeled neurons. D. 40× confocal image showing PHAL axons making close contacts (arrows) with several CTb labeled FoxP2-ir neurons.

Figure 6

Double tract-tracing experiment to demonstrate that AP neurons project to FoxP2-ir neurons of the PBel-inner that send efferent connections to the paraventricular hypothalamic nucleus (PVH). A. A CTb injection (blue) was made into the caudal PVH, which spread into the lateral parvicellular, medial parvicellular, and ventral parts. B. PHAL was injected (red) into the area postrema (AP). C. A schematic line drawing of the parabrachial nucleus (PB) showing the relative positions of FoxP2-ir CTb-labeled PBel-inner neurons that project to the PVH as shown in D–F. The labeled neurons (blue dots) were positioned relative to the superior cerebellar peduncle (scp) using photoimages taken with both darkfield and fluorescence optics. D–F. 40× confocal images showing PHAL axons making close contacts (arrows) with three neurons in the PBel-inner. Other abbreviations: AHA= anterior hypothalamic area; f= fornix; KF= Kölliker-Fuse nucleus; PBcl= central lateral parabrachial subnucleus; PBdl= dorsal lateral parabrachial subnucleus; PBel-outer= external lateral parabrachial subnucleus- outer division; PBem= external medial parabrachial subnucleus; PBm= medial parabrachial subnucleus; PBv= ventral lateral parabrachial subnucleus; scp= superior cerebellar peduncle.

Figure 7

Summary drawings showing A) the AP projects to the HSD2 neurons of the NTS as well as to both the pre-LC and PBel-inner of the dorsolateral pontine region, and B) HSD2 neurons project directly to the pre-LC and PBel-inner. The AP is a circumventricular organ and functions as a chemosensor. Based on c-Fos studies in rats, HSD2 neurons have been implicated along with pre-LC and PBel-inner neurons as components in a neural system that detects peripheral sodium deprivation, signaling the “need” to find and ingest sodium to forebrain areas (Geerling et al., 2006; Geerling and Loewy, 2007). Once sodium ingestion has occurred and the need for sodium has been fulfilled, an inhibitory response that “too much” sodium has been consumed will likely occur. This may be mediated through the AP which provides an inhibitory effect on the HSD2 neurons as well as neurons in the pre-LC and PBel-inner. Lesions of the AP remove this inhibition, and as a consequence, rats consume considerably more amounts of sodium chloride than do normal rats (Curtis et al., 1999). In contrast, HSD2 neurons receive a convergence of both neural and chemosensitive inputs. The former originate from the stomach and duodenum (Shin and Loewy, 2009) and the latter comes from the area postrema (Sequeira et al., 2006). The HSD2 cells are the first central neurons in a pathway that regulates sodium appetite (Geerling et al., 2006), and their first ascending targets are the pre-LC and PBel-inner neurons of the dorsolateral pons. The HSD2 neurons are thought to be a glutamate-containing (Geerling et al., 2008). The efferent projections of the pre-LC and PBel-inner are unknown, except for two reports showing that the PBel projects to the lateral part of the central nucleus of the amygdala (Bernard et al., 1993) and to the dorsolateral and ventral parts of the bed nucleus of the stria terminalis (Alden et al., 1994). Although not defined in this drawing, the HSD2 neurons project to several forebrain sites including the parasubthalamic nucleus, lateral hypothalamic area, paraventricular hypothalamic area, central nucleus of the amygdala, and bed nucleus of the stria terminalis (Geerling and Loewy, 2006; Shin et al., 2008).