Delivery of the 5-HT2A Receptor Agonist, DOI, Enhances Activity of the Sphincter Muscle during the Micturition Reflex in Rats after Spinal Cord Injury

Abstract

Traumatic spinal cord injury (SCI) interrupts spinobulbospinal micturition reflex pathways and results in urinary dysfunction. Over time, an involuntary bladder reflex is established due to the reorganization of spinal circuitry. Previous studies show that manipulation of serotonin 2A (5-HT_2A) receptors affects recovered bladder function, but it remains unclear if this receptor regulates the activity of the external urethral sphincter (EUS) following SCI. To elucidate how central and peripheral serotonergic machinery acts on the lower urinary tract (LUT) system, we employed bladder cystometry and EUS electromyography recordings combined with intravenous or intrathecal pharmacological interventions of 5-HT_2A receptors in female SCI rats. Three to four weeks after a T10 spinal transection, systemic and central blockage of 5-HT_2A receptors with MDL only slightly influenced the micturition reflex. However, delivery of the 5-HT_2A receptor agonist, DOI, increased EUS tonic activity and elicited bursting during voiding. Additionally, subcutaneous administration of DOI verified the enhancement of continence and voiding capability during spontaneous micturition in metabolic cage assays. Although spinal 5HT_2A receptors may not be actively involved in the recovered micturition reflex, stimulating this receptor subtype enhances EUS function and the synergistic activity between the detrusor and sphincter to improve the micturition reflex in rats with SCI.

Keywords: electromyogram; external urethral sphincter; micturition; serotonin; spinal cord injury.

Figure 1

Spinal 5-HT_2A receptor expression remains unchanged after SCI. (A), Western blots show that there is no difference between naïve and SCI rats in terms of protein expression levels of the 5-HT_2A receptor above (T4/5) and below the level of injury (L1/2, L6/S1) (Student’s t-test, all p > 0.05). (B,C), Quantitative PCR analysis reveals that mRNA levels of this receptor were not altered at any of the aforementioned segments in SCI rats (all p > 0.05). GAPDH served as a control.

Figure 2

Intravenous (i.v.) administration of DOI to stimulate 5-HT_2A receptors improves the micturition reflex during bladder cystometry and sphincter EMG in SCI rats. (A), Stimulating 5-HT_2A receptors with the middle dose of DOI (20 µg/kg) prolongs the VI (p < 0.05, Friedman’s test followed by Dunn’s) between voiding contractions (asterisks). The high dose of DOI (0.1 mg/kg) increases EUS tonic activity in the filling phase (p < 0.01). Ensuing injection of MDL (0.1 mg/kg) eliminates the provoked effects. (B), Representative traces show no EUS bursting activity during voiding when saline is delivered. However, the middle dose of DOI triggers EUS bursting activity during voiding. Injection of MDL following the high dose of DOI masks the triggered bursting EUS activity during voiding.

Figure 3

Intrathecal (i.t.) administration of DOI to stimulate 5-HT_2A receptors affects the micturition reflex during bladder cystometry and sphincter EMG in SCI rats. (A), Representative tracers show that stimulating 5-HT_2A receptors with the middle (2 µg/kg) and high (10 µg/kg) doses of DOI prolongs the VI (both p < 0.05, Friedman’s test followed by Dunn’s) between voiding contractions (asterisks) and induces an increase in EUS tonic activity. (B), Importantly, the middle dose of DOI increases the duration of EUS bursting that consists of more regularly occurring active and silent periods along with bladder HFO, indicating an enhancement of detrusor-sphincter coordination during voiding.

Figure 4

Intrathecal (i.t.) administration of MDL abolishes DOI induced excitatory effects on the EUS reflex in SCI rats. A representative cystometry and EMG trace demonstrates that MDL, a 5-HT_2A receptor antagonist (10 µg/kg), shortens the VI (p < 0.05, Friedman’s test followed by Dunn’s) (A) and masks bursting EUS activity (B), leading to the occurrence of detrusor-sphincter dyssynergia.

Figure 5

Stimulating 5-HT_2A receptors with DOI delivery improves spontaneous micturition function in SCI rats. Representative traces show volume-frequency patterns of urination in metabolic cage assays after 5-HT_2A receptor stimulation with DOI within a 6-h time period. Saline injections served as a control. In each “step-like” curve of these traces (A), the vertical lines represent the VV per void and the horizontal lines illustrate the VI. After s.c. injection of the 5-HT_2A receptor agonist DOI (60 µg/kg, 300 µL), (B) there was a significant increase in the VV per void and VI in comparison to saline controls (Paired t-test, all * p < 0.05).