N-methyl-D-aspartate receptor subunit phenotypes of vagal afferent neurons in nodose ganglia of the rat

Abstract

Most vagal afferent neurons in rat nodose ganglia express mRNA coding for the NR1 subunit of the heteromeric N-methyl-D-aspartate (NMDA) receptor ion channel. NMDA receptor subunit immunoreactivity has been detected on axon terminals of vagal afferents in the dorsal hindbrain, suggesting a role for presynaptic NMDA receptors in viscerosensory function. Although NMDA receptor subunits (NR1, NR2B, NR2C, and NR2D) have been linked to distinct neuronal populations in the brain, the NMDA receptor subunit phenotype of vagal afferent neurons has not been determined. Therefore, we examined NMDA receptor subunit (NR1, NR2B, NR2C, and NR2D) immunoreactivity in vagal afferent neurons. We found that, although the left nodose contained significantly more neurons (7,603), than the right (5,978), the proportions of NMDA subunits expressed in the left and right nodose ganglia were not significantly different. Immunoreactivity for NMDA NR1 subunit was present in 92.3% of all nodose neurons. NR2B immunoreactivity was present in 56.7% of neurons; NR2C-expressing nodose neurons made up 49.4% of the total population; NR2D subunit immunoreactivity was observed in just 13.5% of all nodose neurons. Double labeling revealed that 30.2% of nodose neurons expressed immunoreactivity to both NR2B and NR2C, whereas NR2B and NR2D immunoreactivities were colocalized in 11.5% of nodose neurons. NR2C immunoreactivity colocalized with NR2D in 13.1% of nodose neurons. Our results indicate that most vagal afferent neurons express NMDA receptor ion channels composed of NR1, NR2B, and NR2C subunits and that a minority phenotype that expresses NR2D also expresses NR1, NR2B, and NR2C.

Figure 1

Photomicrographs of nodose ganglion neurons labeled with affinity-purified polyclonal primary antisera raised in goat against the NR1 (A), NR2B (B), NR2C (C), and NR2D (D) NMDA receptor subunits. Panels on the left (A, C) show stained neurons in longitudinal sections (20 μm) from the cranial (A,C) and caudal (B,D) portions of nodose ganglia. Scale bar = 50 μm.

Figure 2

Histogram showing the percentage of nodose neurons that express NMDA receptor subunit phenotypes.

Figure 3

Longitudinal serial sections from the same nodose ganglion, separated by 40 μm, stained with affinity-purified polyclonal primary antisera raised in goat against the NR1 (A), and NR2D (B) NMDA receptor subunits. Note that NR1 and NR 2D-labeled neurons are fairly evenly distributed throughout the ganglia, whereas in some instances NR2C-IR perikarya were gathered into clusters of cells (C). Scale bar = 200 μm.

Figure 4

Photomicrographs of nodose ganglion sections double-labeled for NR2B (A) and NR2C (B) NMDA receptor subunits with primary antisera raised in rabbit (NR2B) or goat (NR2C). Double labeling immunofluorescence revealed co-localization of NR2B and NR2C subunit containing neurons (short arrows). Additionally, we found a subpopulation of NR2B subunit-containing neurons that did not express NR2C immunoreactivity (arrowheads) and NR2B immunonegative neurons expressing NR2C immunoreactivity (long arrows). Scale bar = 50 μm.