Gustatory terminal field organization and developmental plasticity in the nucleus of the solitary tract revealed through triple-fluorescence labeling

Abstract

Early dietary sodium restriction has profound influences on the organization of the gustatory brainstem. However, the anatomical relationships among multiple gustatory nerve inputs have not been examined. Through the use of triple-fluorescence labeling and confocal laser microscopy, terminal fields of the greater superficial petrosal (GSP), chorda tympani (CT), and glossopharyngeal (IX) nerves were visualized concurrently in the nucleus of the solitary tract (NTS) of developmentally sodium-restricted and control rats. Dietary sodium restriction during pre- and postnatal development resulted in a twofold increase in the volume of both the CT and the IX nerve terminal fields but did not affect the volume of the GSP terminal field. In controls, these nerve terminal fields overlapped considerably. The dietary manipulation significantly increased the overlapping zones among terminal fields, resulting in an extension of CT and IX fields past their normal boundaries. The differences in terminal field volumes were exaggerated when expressed relative to the respective NTS volumes. Furthermore, increased terminal field volumes could not be attributed to an increase in the number of afferents because ganglion cell counts did not differ between groups. Taken together, selective increases in terminal field volume and ensuing overlap among terminal fields suggest an increased convergence of these gustatory nerve terminals onto neurons in the NTS. The genesis of such convergence is likely related to disruption of cellular and molecular mechanisms during the development of individual terminal fields, the consequences of which have implications for corresponding functional and behavioral alterations.

Figure 1

The anatomical organization of the peripheral gustatory system and the first central synaptic relay in the nucleus of the solitary tract (NTS). The chorda tympani (CT) nerve innervates taste buds in fungiform and foliate papillae on the anterior tongue. The greater superficial petrosal (GSP) nerve innervates taste buds in the nasoincisor duct, the geschmacksstreifen (GS), and the soft palate. Both nerves comprise the VIIth cranial nerve and have cell bodies in the geniculate ganglia. The glossopharyngeal (IX) nerve innervates taste buds in foliate and circumvallate papillae on the posterior tongue, and has cell bodies in the petrosal ganglia. All three nerves terminate bilaterally in the NTS; the glossopharyngeal nerve is shown on the left side only for illustrative purposes. The respective fluorescent markers used to detect each terminal field are noted in parentheses. See methods for a detailed description.

Figure 2

Fluorescent photomicrographs of separated channels of the GSP (green: A, D), CT (red: B, E), and IX (blue: C, F) terminal fields in the dorsal zone of NTS (perimeter marked by dotted line) in control (A–C) and sodium-restricted (D–F) rats. Note the enlarged appearance of the CT and IX terminal fields in sodium-restricted rats compared to controls. See figure 3B, F for corresponding merged images of these separate channels. R, rostral; L, lateral. Scale bar in A equals 100 μm.

Figure 3

Fluorescent photomicrographs comparing the triple labeled terminal fields in horizontal sections of the NTS (approximate perimeter marked by white line) in a control (A–D) and a sodium-restricted (E–H) rat. The CT and IX terminal fields in the dorsal zone of restricted rats (E & F) are more expansive than controls (A & B). (Sections A and E are dorsal to B and F.) In the intermediate zone, the appearance of terminal fields in sodium-restricted rats (G) is also different from controls (C). Often, the IX terminal field in restricted rats extends into the ventral zone (H); however, this does not occur in controls (D). Retrogradely labeled salivatory nuclei are indicated by white arrows (B–D, G & H). Panels I – L show myelin-stained tissue from a separate control rat illustrating the shape of the NTS and other brainstem structures at the respective dorsal to ventral level to the fluorescent images in the same row. The NTS is outlined in black and black arrows point to the solitary tract. Refer to the color key in A to identify individual fields and respective overlap among different terminal fields. R, rostral; L, lateral. Scale bar in E equals 200 μm and refers to panels A – H. Scale bar in I equals 1 mm and refers to panels I – L. 4V, fourth ventricle; 12, hypoglossal nucleus; DCN, dorsal cochlear nucleus; icp, inferior cerebellar peduncle; NTS, nucleus of the solitary tract; Sp5, spinal trigeminal nucleus; sp5, spinal trigeminal tract; VCN, ventral cochlear nucleus

Figure 4

Mean (± SEM) total terminal field volumes of the GSP, CT, and IX nerves and corresponding overlap (e.g., GSP-CT) in the NTS of control (solid bars) and sodium-restricted (open bars) rats. In restricted rats, the CT and IX fields are larger than that of controls. This expansion results in corresponding increases in the overlap among terminal fields. GSP field size does not change. Asterisks indicate p < 0.05.

Figure 5

Zonal distribution of mean (± SEM) terminal field volumes of the GSP, CT, and IX nerves and corresponding overlap in the NTS of control (solid bars) and restricted (open bars) rats. A: Dorsal zone. The largest proportion of the respective terminal fields is contained in the dorsal zone. Also, the greatest increases in terminal field volume occur in this zone in restricted rats compared to controls. B: Intermediate zone. Terminal field volumes are distributed differently in this zone compared to others. Dietary manipulation affects only the IX field and resulting overlap of the IX field with CT and GSP. C: Ventral zone. This zone contains the least volume of terminal fields. The significance in CT-GSP overlap in restricted rats is attributed to a difference in how the GSP and CT fields are distributed within the NTS. Asterisks indicate p < 0.05.

Figure 6

Coronal section through the “intermediate” zone of the rat medulla in a control (A – F) and in a sodium-restricted rat (G – L) showing the terminal field for the GSP (A, G), CT (B, H), and the IX nerves (C, I). Panels D and J show the merged terminal fields and panels E and K show the merged image superimposed on an image of the brainstem obtained through the transmitted light channel on the confocal laser microscope system. Panels F and L show nissl-stained tissue illustrating the location of the NTS relative to other brainstem structures. Dark lines in E, F, K, and L demarcate the approximate boundaries of the subdivisions in the NTS as described by Halsell et al., 1996. These examples illustrate the primary findings of the data obtained in horizontal sections: GSP terminal fields are similar between control (A) and sodium-restricted rats (G), whereas the CT terminal field in sodium-restricted rats (H) extends more dorsally and caudally than in controls (B), and the IX terminal field is enlarged and extends more ventrally in sodium-restricted rats (I) than in controls (C). Importantly, the expanded CT and IX fields in sodium-restricted rats do not invade additional subdivisions in the NTS than in controls (E, K). Note that the borders of the subdivisions are close approximations of what the borders are as revealed by more extensive staining procedures (see Halsell et al., 1996). Panels F and L show that these sections are from the “intermediate” zone and are from approximately the same rostral-caudal level. Refer to the color key in D to identify individual fields and respective overlap among different terminal fields. D, dorsal; L, lateral; T, solitary tract; M, medial subdivision of the NTS, RC, rostral central subdivision of the NTS; V, ventral subdivision of the NTS; RL, rostral lateral subdivision of the NTS. 4V, fourth ventricle; 10, dorsal motor nucleus of the vagus; 12, hypoglossal nucleus; MVe, medial vestibular nucleus; sp5, spinal trigeminal tract; SpVe, spinal vestibular nucleus. Scale bar in A equals 200 μm and applies to A – D and G – J. Scale bar in E equals 200 μm and also applies to panel K. Scale bar in F equals 500 μm and also applies to panel L.

Figure 7

A. Total mean volume of the right half of the NTS in control (solid bars) and restricted (open bars) rats. The NTS volume of restricted rats is significantly smaller than controls. B. Mean volume of GSP, CT, and IX terminal fields and their respective overlapping portions normalized to total NTS volume in control (solid bars) and restricted (open bars) rats. A greater proportion of the NTS is occupied by overlapping terminal fields in restricted rats compared to controls. Error bars denote SEM; asterisks indicate p < 0.05.

Figure 8

Mean (± SEM) number of geniculate (GSP, CT) and petrosal (IX) ganglion cells in control (solid bars) and restricted (open bars) rats. The number of cells in each respective ganglia are similar in control and restricted rats. Note: petrosal ganglia contain 3X more cells than geniculate ganglia.

Figure 9

Model of terminal field organization through the dorsal-ventral extent of horizontal sections from the right NTS in control and restricted rats. Overlapping fields are represented at four levels along the dorsal-ventral axis. See results for details of the overlap among the three fields and a comparison of diet-related differences. Refer to color key to identify individual fields and respective overlap among different terminal fields. Note: Due to the orientation of the NTS within the brainstem, the term “dorsal” sections refers to dorsal-caudal and “ventral” refers to ventral-rostral.