Transsynaptic tracing to dissect supraspinal serotonergic input regulating the bladder reflex in rats

Abstract

Aims: This study was designed to determine specific cell groups of the raphe nuclei (RN) that give rise to supraspinal serotonergic projections regulating the bladder reflex.

Methods: Anesthetized rats underwent surgery to open the abdomen and expose the bladder. A total of 6 µL transsynaptic neuronal tracer pseudorabies virus (PRV-152), encoding for green fluorescent protein (GFP), was injected into the bladder detrusor. After 72 or 96 h, animals were perfused and the brain was dissected for processing transverse and sagittal sections. Subsequently, fluorescent immunohistochemistry for GFP and Serotonin (5-hydroxytryptamine [5-HT]) was performed in the brain sections. Under the microscope, each RN subset was characterized individually from caudal to rostral according to the atlas. GFP⁺ or GFP/5-HT double labeled neurons in each subset were quantified for statistical analysis.

Results: At 72-h post-infection, very few GFP⁺ or GFP/5-HT double-labeled neurons appeared in the brainstem and beyond. In contrast, many labeled neurons were found at these levels after 96 h. Quantitative analysis showed that the majority of infected 5-HT⁺ neurons were located in the pallidus, obscurus, and magnus nuclei. Conversely, very few infected neurons were found in other raphe subsets, that is the pontis, median, dorsal, or linear nuclei. Overall, the raphe magnus had the highest number of GFP-labeled and GFP/5-HT double-labeled cells.

Conclusions: The caudal subsets of RN, especially the raphe magnus, are the main sources of serotonergic input to the lower spinal cord controlling bladder activity.

Keywords: detrusor; pseudorabies virus; retrograde tracing; serotonin; urinary.

FIGURE 1

Pseudorabies virus (PRV)-labeled serotonergic neurons in the coronal sections of the brain. At 96 h after injection of PRV into the bladder wall, immunostaining demonstrates that GFP-labeled serotoninergic (5-HT⁺) neurons are detected in the brainstem and midbrain. A-E, From the rostral to caudal, these double-labeled neurons are shown in each subset of the RN in line with the location of nucleus in the map. The majority of PRV-labeled serotonergic neurons emerge in the medullary parts of raphe nuclei, including the RPa, ROb, and RMg (B1-B3 cell groups). Additionally, some GFP-labeled neurons are 5-HT⁻, suggesting the involvement of non-serotonergic raphe neurons in bladder control. The small image in D shows higher magnification of GFP/5-HT double labeled cells (RPa, raphe pallidus; ROb, raphe obscurus; RMg, raphe magnus; RPn, raphe pontis; MnR, median raphe; DR, dorsal raphe; CLiR, caudal linear raphe; RLiR, rostral linear raphe; Aq, aquaduct; 4V, the 4th ventricle)

FIGURE 2

The sagittal view of the raphe nuclei subsets and the distribution of pseudorabies virus (PRV)-labeled serotonergic (5-HT⁺) cells at 96 h post to infection. Unlike very few GFP-labeled 5-HT⁺ neurons in the rostral cell groups of raphe nuclei, most double immunolabeled cells are mainly present in the caudal subsets. Understanding the labeling pattern in both transverse and sagittal planes renders better clarification of cell distribution in 3-dimensions. Overall, the result of PRV-tracing suggests a complex network of serotonergic neurons modulating the bladder reflex (RPa, raphe pallidus; ROb, raphe obscurus; RMg, raphe magnus; RPn, raphe pontis; MnR, median raphe; DR, dorsal raphe; LiR, linear raphe)

FIGURE 3

GFP-labeled or GFP/serotonin (5-HT) double-labeled neurons are quantified in each subset of the raphe nuclei. A and B, Statistical analysis indicates significantly more (A) GFP-labeled or (B) GFP/5-HT double-labeled neurons in the caudal raphe nuclei at 96-hr post-infection than 72 h, including the RPa, ROb, and RMg. However, very few GFP-labeled or GFP/5-HT double-labeled neurons were detected in the rostral raphe nuclei, that is the RPn, MnR, DR, and LiR, at either 72 or 96 h post-infection. C, High percentage of GFP-labeled 5-HT⁺ to the total of 5-HT⁺ neurons is detected in the 3 caudal raphe nuclei (Unpaired Student’s t-test, *P < 0.05, **P < 0.01; RPa, raphe pallidus; ROb, raphe obscurus; RMg, raphe magnus; RPn, raphe pontis; MnR, median raphe, DR, dorsal raphe; LiR, linear raphe)