Normal urinary bladder function is dependent on the activation of peripheral sensory afferent nerves embedded within the bladder wall. Bladder afferents travel in the splanchnic and pelvic nerves that terminate in the thoracolumbar (TL) and lumbosacral (LS) regions of the spinal cord respectively. Afferent signals generated in the bladder feed into central nervous system circuits to evoke sensations and maintain continence. These afferents express a variety of receptors and ion channels to detect changes in their environment, including bladder stretch. Previous studies have characterised the involvement of individual receptors/ion channels in bladder sensation, but few have looked at the co-expression of these important sensory targets in individual bladder innervating neurons. 

This study aimed to determine the co-expression of receptors/ion channels known to be important in bladder sensation and function, including TRPV1, TRPV4, TRPA1, P2X3, 5-HT3, H1R and M2 within the cell bodies of bladder-innervating neurons. Furthermore, we aimed to determine if these ion channels and receptors are differentially expressed in the cell bodies of nerves travelling in the pelvic and splanchnic nerves.

This study used C57BL/6J female mice (N=12) aged 10-14 weeks old that were randomly assigned to experimental procedures. Mice underwent retrograde neuronal tracing from the bladder wall using Cholera Toxin subunit B conjugated with AlexaFluor 488. 4 days later, dorsal root ganglia (DRG), where the cell bodies of peripheral afferents are contained, were surgically removed from the TL (T10-L1) and LS (L5-S1) regions of the spinal cord. 

Isolated LS and TL DRGs underwent cell culture procedures to obtain a single-cell suspension of sensory neuron cell bodies. The suspensions were plated on coverslips and left to incubate for 4hrs for cell picking or 18-32hrs for calcium imaging. Successfully traced cells could then be identified by their green fluorescence under a fluorescent microscope.

Receptor/ion channel mRNA expression and co-expression was determined using single-cell RT-PCR. Bladder-innervating DRG neurons in cell culture were isolated through single-cell picking and immediately lysed in a lysis buffer containing DNase. The lysed cells were placed in dry ice and then stored at -80°C until cDNA synthesis was performed. Expression and co-expression of each target receptor was identified within individual cells (LS neurons: 32; TL neurons: 15) using commercially available hydrolysis probes (TaqMan; Life Technologies).  

Functional co-expression of our target receptors and ion channels was determined using live-cell calcium imaging of bladder-innervating neurons in response to agonists (GSK1016790A 100μM – TRPV4, 5-HT 100μM – 5-HT3, Histamine 100μM – H1R, Carbachol 100μM – M2, ATP 100μM – P2X3, AITC 300μM – TRPA1 and Capsaicin 50nM – TRPV1). To perform calcium imaging, DRG neuronal cell cultures were incubated with the intracellular calcium indicator Fura-2 AM. Cell responses were identified as an increase in fluorescence intensity in response to a specific agonist. Responses and co-responses of bladder traced neurons (LS neurons: 46; TL neurons: 37) were recorded using Metafluor (Molecular Devices).

Single-cell RT-PCR revealed that more than 80% of bladder-innervating LS neurons expressed Htr3a, P2rx3 and Trpv1 (Figure 1), additionally, 75% of LS neurons co-expressed these 3 gene targets. Less than 45% of LS neurons expressed Hrh1, Chrm2, Trpa1 and Trpv4. For the bladder-innervating TL neurons, more than 80% of the cells expressed Htr3a, P2rx3, Trpa1 and Trpv1 (Figure 1), co-expression of these 4 receptors was also observed in 67% of TL neurons. Hrh1 and Chrm2 were expressed by 40% of TL neurons and no cells expressed Trpv4. Percentage expression of each target receptor was compared between the bladder LS and TL DRG neurons using the Chi-squared test. A significant difference was identified between the percentage of neurons expressing Trpa1 (p < 0.001), where bladder-innervating TL DRG neurons had a significantly higher proportion of cells expressing Trpa1 compared to LS DRG neurons (Figure 1). 

Calcium imaging experiments showed that more than 70% of bladder-innervating LS neurons responded to 5-HT, ATP and Capsaicin, while approximately half of the cells responded to AITC and significantly fewer neurons responded to Histamine, Carbachol and GSK (Figure 2). We observed that 41% of LS neurons co-responded to 5-HT, ATP and Capsaicin. Different functional responses were observed in TL DRG, where more than 40% of bladder-innervating TL DRG neurons responded to ATP and AITC, and around 40% of neurons co-responded to both receptor agonists. Less than 15% of TL neurons responded to 5-HT and Histamine, and no cells responded to GSK and Carbachol. Further Chi-squared analysis compared the responses made by bladder-innervating LS and TL DRG neurons to each agonist. Overall, responses to 5-HT (p < 0.0001), ATP (p < 0.01) and Capsaicin (p < 0.01) occurred in a significantly higher number of LS neurons compared to the TL neurons (Figure 2).

This study highlighted, for the first time, key differences in the molecular and functional expression of receptors in the cell bodies of bladder-innervating pelvic and splanchnic nerves. We were able to discern distinct patterns of receptor expression and co-expression within neurons from LS and TL DRG. The differences in receptor expression and responses we observed suggest that bladder pelvic and splanchnic nerves may have distinct functions within the bladder. The significantly greater functional responses of LS DRG to 5-HT and ATP suggest these neurons may be more responsive to neurotransmitters released from the urothelium (ATP) and immune cells (5-HT). These two sensory pathways may mediate transduction of distinct sensations arising from the bladder. 

The lack of calcium imaging responses to Muscarinic or TRPV4 agonism suggest that neither TRPV4, nor muscarinic receptors are directly involved in bladder afferent signalling.

Our research has shown that there is a high level of functional and molecular co-expression of sensory receptors within bladder-innervating LS and TL DRG. Furthermore, significant differences exist in the expression and function of sensory receptors within the cell bodies of pelvic and splanchnic bladder-innervating nerves. Further investigation into the relationship between these receptors will lead to an improved understanding of the mechanism underlying bladder sensation.