Localization of peripheral autonomic neurons innervating the boar urinary bladder trigone and neurochemical features of the sympathetic component

Abstract

The urinary bladder trigone (UBT) is a limited area through which the majority of vessels and nerve fibers penetrate into the urinary bladder and where nerve fibers and intramural neurons are more concentrated. We localized the extramural post-ganglionic autonomic neurons supplying the porcine UBT by means of retrograde tracing (Fast Blue, FB). Moreover, we investigated the phenotype of sympathetic trunk ganglion (STG) and caudal mesenteric ganglion (CMG) neurons positive to FB (FB+) by coupling retrograde tracing and double-labeling immunofluorescence methods. A mean number of 1845.1±259.3 FB+ neurons were localized bilaterally in the L1-S3 STG, which appeared as small pericarya (465.6±82.7 µm2) mainly localized along an edge of the ganglion. A large number (4287.5±1450.6) of small (476.1±103.9 µm2) FB+ neurons were localized mainly along a border of both CMG. The largest number (4793.3±1990.8) of FB+ neurons was observed in the pelvic plexus (PP), where labeled neurons were often clustered within different microganglia and had smaller soma cross-sectional area (374.9±85.4 µm2). STG and CMG FB+ neurons were immunoreactive (IR) for tyrosine hydroxylase (TH) (66±10.1% and 52.7±8.2%, respectively), dopamine beta-hydroxylase (DβH) (62±6.2% and 52±6.2%, respectively), neuropeptide Y (NPY) (59±8.2% and 65.8±7.3%, respectively), calcitonin-gene-related peptide (CGRP) (24.1±3.3% and 22.1±3.3%, respectively), substance P (SP) (21.6±2.4% and 37.7±7.5%, respectively), vasoactive intestinal polypeptide (VIP) (18.9±2.3% and 35.4±4.4%, respectively), neuronal nitric oxide synthase (nNOS) (15.3±2% and 32.9±7.7%, respectively), vesicular acetylcholine transporter (VAChT) (15±2% and 34.7±4.5%, respectively), leu-enkephalin (LENK) (14.3±7.1% and 25.9±8.9%, respectively), and somatostatin (SOM) (12.4±3% and 31.8±7.3%, respectively). UBT-projecting neurons were also surrounded by VAChT-, CGRP-, LENK-, and nNOS-IR fibers. The possible role of these neurons and fibers in the neural pathways of the UBT is discussed.

Figure 1.

Western blot immunolabeling of tyrosine hydroxylase, dopamine beta-hydroxylase, somatostatin, neuropeptide Y and vesicular acetylcholine transporter in porcine dorsal root ganglia, sympathetic trunk ganglia and spinal cord. The number on the left of each line indicates the molecular weight. The images of the different immunoblots were slightly adjusted in brightness and contrast to provide a uniform background. TH: tyrosine hydroxylase; DβH: dopamine beta-hydroxylase; NPY: neuropeptide; VAChT: vesicular acetylcholine transporter.

Figure 2.

Histogram showing the frequency distribution of urinary bladder trigone-projecting neurons (mean total number 1845.1±259.3, n=4) in each segmental level of the sympathetic trunk ganglia.

Figure 3.

A) Photomicrographs of the longitudinal section of the L7 right sympathetic trunk ganglion (STG L7), one of the ganglia containing the highest density of Fast Blue (FB)-labeled cells innervating the pig urinary bladder trigone. A1) Distribution of the FB-labeled sympathetic postganglionic neurons at the ganglion periphery as visible at low magnification (1x); the white rectangle indicates the area where the image shown in A2 has been taken from. A2) Morphology of the sympathetic postganglionic neurons labeled, as visible at higher magnification (20x). B) Photomicrographs of the longitudinal section of the right caudal mesenteric ganglion (CMG). B1) Distribution of the FB-labeled sympathetic postganglionic neurons at the ganglion periphery as visible at low magnification (1x); the white rectangle indicates the area where the image shown in B2 has been taken from. B2) Morphology of the same labeled neurons as visible at higher magnification (20x). C) Photomicrographs of a section of one micro-ganglion of the pelvic plexus (PP) showing the distribution of the postganglionic FB-labeled neurons (C1). C2) Morphology of the PP labeled neurons as visible at higher magnification (20x).

Figure 4.

Mean sizes (±SEM, n=4 pigs) of urinary bladder trigone-projecting post-ganglionic neurons in the sympathetic trunk ganglia (STG), caudal mesenteric ganglia (CMG) and pelvic ganglia (PP). The soma cross sectional area was measured on at least two hundred FB-labeled cells from each neuronal class in each animal. One-way ANOVA analysis revealed significant differences (P<0.001) between the mean sizes of neurons in the three kind of ganglia. Asterisks in the figure indicate a positive significance level (*P<0.05) obtained in the comparison of PP vs CMG and STG with Tukey post-hoc test.

Figure 5.

Histograms showing the mean ± SEM percentages (n=4 pigs) of sympathetic trunk ganglia (STG) and caudal mesenteric ganglia (CMG) neurons projecting to the pig UBT (FB+ neurons) immunoreactive (+) to each antiserum and the proportion of these FB+ neurons that co-expressed also TH-immunoreactivity (TH+).

Figure 6.

Fluorescence micrographs of longitudinal sections of the porcine sacral (S1) sympathetic trunk ganglion containing FB-positive perikarya double labeled for TH and one of the different markers employed. In the group of figures labeled with the letter A, the arrows indicate three FB-positive perikarya (A1) containing simultaneously DβH (A2) and TH (A3). In the group of figures labeled with the letter B, the arrows indicate three FB-positive cell bodies (B1) double immunolabeled for NPY (B2) and TH (B3). In the group of figures labeled with the letter C, the arrow points to a FB-positive cell body (C1) which simultaneously contained CGRP (C2) and TH (C3). In C2, near this cell, are also visible varicose CGRP-immunolabeled nerve fibers. Finally, in the group of figures labeled with the letter D, the arrow points to a FB-positive cell body (D1) containing simultaneously SOM (D2) and TH (D3).

Figure 7.

Microphotographs of sections from the porcine STG S1 (A1-B4) and CMG (C1-D4). A) the arrowhead shows an UBT-projecting neuron (FB-positive perikarion) (A1) immuno-negative for TH (A3, Texas Red visualisation) and VAChT (A2, FITC visualisation), but surrounded by a meshwork of nerve terminals (indicated by arrows) immunore-active for VAChT (A2, FITC visualisation); these images were digitally superimposed in A4. B) the arrowhead shows a FB-positive neuron (B1) immunoreactive both for LENK (B2, FITC visualisation) and for TH (B3, Texas Red visualisation); single nerve fibres immunoreactive for LENK (FITC visualisation) are visible between the ganglion cells and indicated by arrow. These images were digitally superimposed in B4. C) arrowheads indicate two FB-positive neurons (C1) weakly immunoreactive for CGRP (C2, FITC visualisation) and TH (C3, Texas Red visualisation); single nerve fibres immunoreactive for CGRP (FITC visualisation) are visible between and on the ganglion cells and indicated by the arrow; these images were digitally superimposed in C4. D) arrowheads show three FB-positive neurons simultaneously immunostained for nNOS (D2, FITC visualisation) and TH (D3, Texas Red visualisation); nNOS-immunoreactive fibres (FITC visualisation) surrounding the ganglion cells are indicated by arrows; the images were digitally superimposed in D4 (double-labelled nerve cells are yellow).

Figure 8.

Microphotographs of sections from the porcine CMG containing FB-positive perikarya double labeled for TH and one of the different markers employed. A) Arrows indicate two FB-positive perikarya (A1) that simultaneously contained SP (A2) and TH (A3). B) Arrows indicate two FB-positive cell bodies (B1) double immunolabeled for VIP (B2) and TH (B3). C) Arrow points to a FB-positive cell body (C1) which simultaneously contained VAChT (C2) and TH (C3). D) Arrows indicate two FB-positive cell bodies (D1) which simultaneously contained nNOS (D2) and TH (D3). E) Arrows indicate two FB-positive cell bodies (E1) which simultaneously contained LENK (E2) and TH (E3).