The spinal serotonergic pathways have been reported to be involved in the control of urethral continence reflexes.  Previous study demonstrated that serotonin (5-HT) receptor subtypes, 5-HT1A or 5-HT2C, respectively, reduce or enhance the urethral continence reflex during sneezing in rats [1]. However, because there are other multiple excitatory and inhibitory 5-HT receptor subtypes, the overall effects of the 5-HT system or the subtype-specific mechanism in the control of the urethral function remain to be elucidated.  Moreover, 5-HT7 receptors are shown to be expressed in the spinal cord and dorsal root ganglia of rats [2] although the role of 5-HT7 receptors in the urethral continence reflex is not well elucidated. Therefore, in this study, we examined the impacts of 5-HT depletion induced by p-chlorophenylalanine (PCPA) and the effects of activation of 5-HT receptor subtypes such as 5-HT2C and 5-HT7 on urethral baseline activity and reflex contractions during sneezing in PCPA-pretreated rats.

Sixty adult female Sprague-Dawley rats weighing 228–270 g were used. We measured the amplitude of urethral pressure responses during sneezing (A-URS) and urethral baseline pressure (UBP) using a microtransducer-tipped catheter for 32 rats. We measured sneeze-induced leak point pressure (S-LPP), the lowest pressure value that induced fluid leakage from the urethral orifice for 28 rats.  Pre-drug sneeze-induced responses were measured before intravenous application of a 5-HT2C agonist (CP-809101), a 5-HT2C antagonist (SB-242084), a 5-HT1A antagonist (WAY-100635), a 5-HT7 agonist (LP44), and a 5-HT7 antagonist (SB-269970) in PCPA-pretreated rats. WAY-100635 was administered before LP44 administration to suppress the partial 5-HT1A effect of LP44. Data are expressed as mean ± standard error (SE). Unpaired t-test was used to compare the A-URS, UBP and Pabd between normal and PCPA-pretreated rats. Paired t-test was also used to compare the values before and after CP-809101 administration. One-way analysis of variance followed by Bonferroni’s multiple comparison tests were used to compare before and after administrations of multiple drugs. Statistical significance was set at P values < 0.05.

1)	A-URS and UBP
Two-day pre-treatment of PCPA (n = 5) significantly decreased A-URS from 71.8 ± 7.1 to 36.8 ± 4.3 cmH2O (P < 0.01), and UBP from 31.1 ± 3.0 to 17.8 ± 2.2 cmH2O (P < 0.01) compared to normal rats (n = 6).  In PCPA-pretreated rats, CP-809101 (n = 4) or LP44 with pre-administration of WAY-100635 (5-HT1A antagonist) (n = 7) significantly increased A-URS from 42.1 ± 5.7 to 66.2 ± 6.5 cmH2O (P < 0.01) and 30.0 ± 2.7 to 50.5 ± 5.3 cmH2O (P < 0.01) and UBP from 17.3 ± 1.2 to 32.4 ± 2.7 cmH2O (P < 0.05) and 15.2 ± 1.6 to 26.6 ± 2.1 cmH2O (P < 0.01), respectively (Figure 1). The effects of CP-809101 and LP44 were antagonized by antagonists of each receptor subtype (SB-242084; n-=4 and SB-269970; n=6, respectively). The average values of sneeze-induced increases in Pabd measured by intra-abdominal catheters were not significantly different between normal and PCPA-pretreated rats or after injection of any drugs.
2)	S-LPP
Although SUI was not observed even at the highest intravesical pressure during sneezing of 108.8 ± 5.6 cmH2O in normal rats (n = 6), fluid leakage from the urethra during sneezing was observed with S-LPP values of 40.1 ± 2.5 cmH2O in all PCPA-pretreated rats (n = 6). In these 5-HT-depleted rats, fluid leakage from the urethra was still observed during sneezing after the treatment with CP-809101(n =4) or LP44 (n =6), which, however, significantly increased S-LPP by 28.0 cmH2O; from 39.7 ± 4.5 cmH2O to 67.7 ± 3.1 cmH2O (P < 0.01), and 15.2 cmH2O; from 37.8 ± 1.4 cmH2O to 53.0 ± 3.4 cmH2O (P < 0.01), respectively, compared to rats treated with PCPA alone. The effects of CP-809101 and LP44 were blocked by antagonists of each receptor subtype (n =3 each) (Figure 2).

In present study, we first examined the effect of PCPA because systemic pre-treatment of PCPA for 2 days reportedly resulted in 95 % depletion in brain 5-HT content [3]. Using this method, we clarified that PCPA treatment caused SUI during sneezing, indicating that the overall 5-HT system acts to maintain urinary continence. Furthermore, we examined the effects of 5-HT subtype-selective drugs (5-HT2C and 5-HT7) on urethral baseline activity and reflex contractions during sneezing in rats.

The results of this study indicate that activation of 5-HT receptors, as a whole, enhances the active urethral closure reflex during sneezing and that 5-HT2C and 5-HT7 receptors have facilitatory roles in urethral continence mechanisms in rats.

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