Nakano M et al. (2009), Mediolateral and rostrocaudal topographic organ...

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J Comp Neurol 2009 Mar 20;5133:292-314. doi: 10.1002/cne.21956.

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Mediolateral and rostrocaudal topographic organization of the sympathetic preganglionic cell pool in the spinal cord of Xenopus laevis.

Nakano M , Goris RC , Atobe Y , Kadota T , Funakoshi K .

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The sympathetic preganglionic cell pool in Xenopus laevis can be divided into four parts, i.e., the intercalated nucleus (IC) and the intermediolateral nucleus (IML) located respectively at the medial and the lateral borders of the lateral field, the lateral funiculus, and the ventral field within the thoracolumbar spinal segments. We compared the location of the preganglionic cells labeled following tracer application to the paravertebral sympathetic chain with those labeled following application to the celiac ganglion (CG), the adrenal gland (AG), and the splanchnic nerves (SNs) and found that their relative contribution differs depending on the sites. In tracer application to the paravertebral chain ganglia and the sympathetic trunk, 31.4-41.9% and 43.9-58.4% of labeled cells were detected respectively in the IC and in the IML, whereas application to the CG, AG, and on all the SNs, revealed that more than 84% of labeled cells were found in the IML and in the lateral funiculus with less than 8.6% in the IC. The contribution of the ventral field cells was less than 7.5% in all experiments. This type of topographic cytoarchitecture is a character shared with the mammalian preganglionic cell pool, but what distinguishes it from that of mammals is its systematic form throughout the entire longitudinal extent of the pool. In Xenopus, differences of mean soma areas and dendritic projections of labeled cells also suggest that the cell pools are distinguished not only by their location and axonal projections, but also by the morphology of their cells.

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Species referenced: Xenopus laevis
Genes referenced: chat sia2 th th2

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	Figure 1. The typical organization of the paravertebral sympathetic components and the ventral branches of the spinal nerves (SN2–10) in Xenopus. The basal regions of the skull are cut away (dotted lines) to show the cephalic part of the sympathetic trunk (asterisk). Each paravertebral chain ganglion is referred to in the text by the number of the spinal nerves connected to it by a communicating branch. In Xenopus, there are five splanchnic nerves (Spl1–5) originating from the sympathetic chain and running along the lumbar arteries to terminate on the ventral surface of the dorsal aorta. The dextran application sites are indicated on the right side of the Figure (arrows, a–j). The inset is an enlargement of the boxed area and indicates the dextran application site (c) in that area.
	Figure 2. Prevertebral sympathetic postganglionic cells of Xenopus. The cells are visualized with TH immunohistochemical staining. A: Confocal image from a single 1-m optical section of a transverse section through the celiaco-mesenteric (CA) and mesenteric (MA) arteries demonstrating the distribution and morphology of the celiac ganglion cells. B: Confocal images from a single 1-m optical section of a horizontal section of the dorsal aorta (DA) in the vicinity of the origin of the celiaco-mesenteric artery (B1) and of the common iliac artery (B2). Twin arrows indicate clusters of immunoreactive postganglionic cells within the adventitia of the aorta. The insets in A and B are higher magnifications, demonstrating the monopolar shape of the immuno- reactive cells. They have only one neuronal process, the axon (arrow- heads). Scale bar 100 m in B2 (also applies to A,B1); 20 m in inset of B2 (applies to all insets).
	Figure 3. Western blot analysis of proteins from the celiac ganglion (A), the paravertebral sympathetic ganglia (B), and the thoracic spinal cord (C,E) of Xenopus laevis with anti-TH and anti-ChAT antibodies. Extract of rat thoracic spinal cord (D,F) was used as a positive control. In both Xenopus and rats, the anti-TH antibody recognized a single band at 59 kDa, and the anti-ChAT antibody recognized a single band at 70 kDa, corresponding to the molecular weights predicted for TH and ChAT, respectively. Molecular weight standards are indicated at left (kDa).
	Figure 4. Spinal distribution of sympathetic preganglionic cells labeled after BDA application to the proximal end of the cut communicating branch of the sixth chain ganglion in Xenopus. A: Transverse section of the spinal cord at the level of the sixth ventral root showing the concentrated distribution of labeled cells to the IC and the IML. An arrow indicates the cell type that we call lateral funiculus cells (LFc). Labeled preganglionic cells were also seen in the ventral field (Vfc in B,C). Twin arrows indicate one of the cells labeled in the space between the IC and the IML. Arrowheads indicate labeled primary afferent terminals. The bracketed asterisk indicates the approximate level of reconstructed confocal images in Figure 9. B: Diagram illustrating the procedure used to assign preganglionic cells to different four cell types (see Materials and Methods). C: Average occurrence of labeled preganglionic cells of each cell type (n 3). D: Composite camera lucida drawing of serial horizontal sections of the spinal cord showing the restricted rostrocaudal distribution of BDA-labeled preganglionic cells. Dots represent the labeled cells; because of clustering and superposition of some cells, the number of dots does not represent actual values. Numbers and dotted lines indicate the segmental level of ventral roots. For abbreviations, see list. Scale bar 200 m in A; 500 m in D.
	Figure 5. Distribution of labeled preganglionic cells after BDA application to the paravertebral sympathetic chain ganglia, the second (SG2), the seventh (SG7) and the tenth (SG10) ganglia, and to the sympathetic trunk (ST) between the fourth and the fifth chain ganglia of Xenopus. A: Average occurrence of labeled cells of each type (three frogs each). The great majority of cells are localized in the IC and the IML. B: Average occurrence of labeled cells of each spinal segment (three frogs each). C: Composite camera lucida drawings of serial horizontal sections from spinal cords in the five different experiments. Dots represent labeled preganglionic cells; each dot may represent more than one cell due to superposition of labeled cells. Numbers and dotted lines indicate the segmental level of ventral roots. For abbreviations, see list. Scale bar 1 mm in C.
	Figure 6. Distribution of labeled sympathetic preganglionic cells after BDA application to the celiac ganglion (CG) or after FB application to the left adrenal gland (AG) in Xenopus. A: Average occurrence of labeled cells of each type (three frogs each). B: Average occurrence of labeled cells of each spinal segment (three frogs each). Percentages in A and B were calculated with values from both sides of the spinal cord. C: Photomicrographs of serial horizontal sections (C1,2) at the level of the fifth ventral root, and a composite camera lucida drawing (C3) of serial horizontal sections from the same spinal cord as C1 and C2. White arrows in C1 indicate unlabeled IC cells. D: Composite camera lucida drawing of serial horizontal sections from the spinal cord in an FB experiment on the adrenal gland. Dots and numbers in C3 and D represent the labeled preganglionic cells and the segmental level of ventral roots, respectively. Each dot may represent more than one cell due to superposition of labeled cells. In both tracer applications, the preganglionic cells are largely unlabeled in the cell column of the IC. For abbreviations, see list. Scale bar 200 m in C1,C2; 1 mm in C3,D.
	Figure 7. Distribution of the sympathetic preganglionic cells labeled by BDA application from the first to the fifth splanchnic nerves (Spl1–5) in Xenopus. A: Average occurrence of labeled cells of each type (three frogs each). The predominant distribution of labeled cells to the IML is similar to that seen in experiments on the celiac ganglion and on the adrenal gland (Fig. 6A). B: Average occurrence of labeled cells of each spinal segment (three frogs each). C: Composite camera lucida drawings of serial horizontal sections from spinal cords in each experiment on the nerves. Dots represent labeled preganglionic cells; each dot may represent more than one cell due to superposition of labeled cells. Numbers and dotted lines indicate the segmental level of ventral roots. For abbreviations, see list. Scale bar 1 mm in C.
	Figure 8. Reconstructed confocal images from serial optical sections of one horizontal section from spinal cords after the simultaneous application of different fluorescent dextran amines to different sympathetic structures in Xenopus, showing the strong axonal projection of IC cells to the second and tenth chain ganglia. A: Reconstruction from seven 5-m serial optical sections at the level of the fifth ventral root after the application of FDA to the first splanchnic nerve (green) and TDA to the sympathetic trunk between the fourth and the fifth sympathetic ganglion (magenta). B: Reconstruction from seven 5-m serial optical sections at the eighth ventral root after the application of FDA to the fourth splanchnic nerve and TDA to the tenth chain ganglion. In both experiment A and experiment B, the preganglionic cells were only labeled by one tracer, either FDA or TDA. Bright white fluorescence within cell columns of the IML and the IC (circled areas in A,B) is due to an overlap of different preganglionic cells labeled with either FDA or TDA. For abbreviations, see list. Scale bar 100 m in A,B.
	Figure 9. ChAT-immunoreactive cell columns in the lateral field of Xenopus thoracolumbar spinal segments. In both A and B, because the approx- imate level of optical sections and the drawing is that of the bracketed asterisk in Figure 4A, cells labeled in the ventral field are not shown. A: Reconstructed confocal images from 11 3-m horizontal serial optical sections from the levels between ventral roots of the fifth and the sixth (A1) and of the sixth and the seventh (A2) and from the level of the eighth ventral root (A3) of one spinal cord. The appearance of the cell columns is considered as representative of the IC and the IML cell columns, but it is also necessary to note that cholinergic inter- neurons may be involved in both the immunoreactive cell columns. Twin arrows indicate small clustered groups of the immunoreactive cells within the cell columns. In A2 and A3, the IML cell column appears as a sparse distribution of cells compared with that of the IC cell column. This may be because the IML cells appear to be also distributed dorsoventrally in the intermediolateral region, and the IC cells are more packed at the lateral border of the lateral field (see Fig. 4A,B, see also Nikundiwe et al., 1982). B: Composite drawing of reconstructed confocal images from serial optical horizontal sections of a different spinal cord from that of A. Dots represent the immuno- reactive cells. As in Figure 7C, each dot may represent more than one cell due to superposition. Numbers indicate the level of ventral roots. The cell columns of the IC and IML have an interconnection with cells distributed in the space between the columns from a level between the fourth and the fifth ventral roots to the level of the eighth ventral root. There are also regional differences in the density of cell distri- bution, especially in the region between the sixth and the seventh ventral roots. For abbreviations, see list. Scale bars 100 m in A1–A3; 400 m in B.
	Figure 10. Percentage histograms of somal areas of labeled IC cells (black bars) and IML cells (white bars) after application of BDA to the second sympathetic ganglion (SG2), the communicating branch of the sixth chain ganglion (CB6), the tenth chain ganglion (SG10), and the celiac ganglion (CG) and after FB application to the adrenal gland (AG) in Xenopus. Left and right columns are respectively the measurements of the transverse and the horizontal planes of sections in different spinal cords from each tracer experiment. The longitudinal and the horizontal axes of all histograms indicate respectively the percentage of cells and the cell body area (m2). Mean soma areas and the number of cells are shown in Table 2. In SG2, CB6, and SG10, the mean soma area for IC cells was larger than that for IML cells in both planes (P < 0.05).
	Figure 11. Dendritic projections of the sympathetic preganglionic cells in Xeno- pus. A: Reconstructed confocal images from 35 1-’m (A1 and A2 each) horizontal serial optical sections from two frozen sections of one spinal cord at the level of the fifth ventral root after TDA application to the sympathetic trunk between the fourth and the fifth chain ganglia. A2 is a section dorsal to A1. The approximate level of optical sections in A2 is that of the bracketed asterisk in C. Rostrocaudal dendritic projections are conspicuous around the region of the IC compared with the projections around the IML. Interconnections between the nuclei with lateral dendrites of IC cells and medial dendrites of IML cells is also seen in encircled areas in A1. Arrowheads in A1 indicate lateral funiculus cells. B: Photomontage of one labeled preganglionic cell of the IC (arrowhead) after BDA application to the cut communi- cating branch, showing the prominent projections of rostral dendrites. C,D: Labeling of contralateral projections of dendrites (single arrows) after BDA application to the seventh chain ganglion (C) or to the sympathetic trunk (D). C: Transverse section at the level of the seventh ventral root. D1–3: Horizontal sections from one spinal cord cut through the region of the blood sinuses (D1), the central canal (D2), and the somatic motoneuron pool (SM; D3) at the level of the fifth ventral root. Asterisks in C indicate blood sinuses. The contralateral projections of dendrites often threaded their way through the septa of the sinuses (arrowheads in C), but sometimes passed through the ventral region of the central canal (D2,3). In addition to the region just medial to the cell column of the IC, dense distribution of labeled dendrites is seen along the lateral edge of the blood sinuses and of the central canal, and in the ventral region of the canal (twin arrows in A2,C,D). Arrowheads in D2 and D3 indicate cells of the IML and of the ventral field, respectively. For abbreviations, see list. Scale bar 50 ’m in B; 100 ’m in A1,A2,C,D1–D3.