Roles of glutamatergic and serotonergic mechanisms in reflex control of the external urethral sphincter in urethane-anesthetized female rats

Abstract

This study was conducted to examine reflex mechanisms that mediate urinary bladder and external urethral sphincter (EUS) coordination in urethane-anesthetized female Sprague-Dawley rats. We investigated the properties of EUS reflexes elicited by electrical stimulation of pelvic nerve afferent axons (pelvic-EUS reflex). The changes in the reflexes induced by bladder distension and administration of agonists or antagonists for glutamatergic or serotonergic receptors were examined. The reflexes consisted of an early response (ER, 18- to 22-ms latency) and a late, long-duration (>100-ms latency) response (LR), which consisted of bursts of activity at 20- to 160-ms interburst intervals. In a few experiments, a reflex with an intermediate (40- to 70-ms) latency was also identified. With the bladder empty, the ER, but not the LR, was detected in the majority of experiments. The LR was markedly enhanced when the bladder was distended. The ER remained, but the LR was abolished, after spinal cord transection at T8-T9. The ER and LR were significantly decreased 75 and 35%, respectively, by the N-methyl-D-aspartate receptor antagonist MK-801 (0.3 mg/kg iv), but only decreased 18 and 14%, respectively, by the alpha-amino-5-methylisoxazole-4-propionate receptor antagonist LY-215490 (3 mg/kg iv). The serotonin (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (1 mg/kg iv) enhanced spontaneous EUS activity and the pelvic-EUS reflex. WAY-100635 (0.1-1 mg/kg iv), a 5-HT1A antagonist, reversed the effect of 8-hydroxy-2-(di-n-propylamino)-tetralin and suppressed EUS activity and the pelvic-EUS reflex. These results indicate that glutamatergic and serotonergic mechanisms are important in the reflex pathways underlying bladder- sphincter coordination in rats.

Fig. 1

Experimental setup. EUS, external urethral sphincter; stim and rec, stimulation and recording electrodes.

Fig. 2

Pelvic EUS reflex was enhanced by bladder distension in spinal cord-intact rats. A: EUS electromyogram (EMG) activity without electrical stimulation (top trace) and pelvic-EUS reflex elicited by a single shock (7.5 V, 1.0 Hz, 0.05-ms pulse width) to the pelvic nerve (●, bottom trace) when the bladder was empty. Reflex responses consisted of a large early response (ER) and small intermediate (IR) and late responses (LR). B: EUS EMG activity without electrical stimulation when the bladder was distended by 0.2 ml of saline (top trace) and the LR of the pelvic-EUS reflex was enhanced by bladder distension (bottom trace). Responses represent average of 10 individual reflexes recorded on a computer.

Fig. 3

Representative examples of individual and averaged pelvic-EUS reflexes when the bladder was distended by 0.5 ml of air. Individual and averaged reflexes were recorded in the same spinal cord-intact animal. A: control spontaneous activity without electrical stimulation (top trace) and a single-sweep recording showing the pelvic-EUS reflex, which consisted of an ER and a LR composed of a series of burst discharges at 100- to 150-ms intervals (bottom trace). Bursts consisted of low-amplitude long-duration firing and one or two high-amplitude transient potentials. B: single-sweep recording showing variability of LR and consistency of ER. C: average of 5 individual recordings showing high-amplitude potentials of LR. D: average of 10 individual recordings showing that high-amplitude potentials of LR did not average well, presumably because of variation in latency between individual recordings. However, low-amplitude and long-duration burst activity is still obvious. ●, Electrical stimulation (8 V, 1.0 Hz, 0.05-ms pulse width).

Fig. 4

Effect of crushing the pelvic nerve peripherally and centrally to the site of stimulation on the pelvic-EUS reflex when the bladder was distended. Each trace represents an individual recording. Individual reflexes were recorded in the same spinal cord-intact animal. A: EUS spontaneous activity without electrical stimulation (top trace) and pelvic-EUS reflex elicited by electrical stimulation (bottom trace). B: some high-amplitude potentials of LR were eliminated by crushing the pelvic nerve peripherally to the site of stimulation. C: pelvic-EUS reflex was completely eliminated when the pelvic nerve was crushed centrally to the site of stimulation, but spontaneous EUS activity remained, because the contralateral pelvic nerve remained intact. ●, Electrical stimulation (6 V, 0.5 Hz, 0.05-ms pulse width).

Fig. 5

Effects of MK-801 and LY-215490 on the pelvic-EUS reflex. Individual reflexes were recorded in the same spinal cord-intact animal. A: pelvic-EUS reflex elicited by a single shock when the bladder was distended. B: reduction in amplitude in the pelvic-EUS reflex after administration of MK-801 (0.3 mg/kg iv). C: after MK-801, LY-215490 (3 mg/kg iv) suppressed the ER. Responses represent individual reflexes recorded on a computer. ●, Electrical stimulation (5 V, 0.5 Hz, 0.05-ms pulse duration).

Fig. 6

Effects of drugs on reflex area of the ER and LR in the pelvic-EUS reflex in spinal cord-intact rats. Area of ER was measured when the bladder was empty; area of the LR was measured when the bladder was distended by 0.2 ml of saline. A: ER and LR were significantly decreased by 75 and 35%, respectively (n = 11, compared with control recordings without drugs) after MK-801 (0.3 mg/kg iv). ER and LR were decreased by 18 and 14%, respectively (n = 7, P > 0.05 vs. control recordings without drugs) after LY-215490 (3.0 mg/kg iv). When MK-801 was administered first, followed 30 min later by LY-215490, ER and LR were reduced by 87 and 53%, respectively (n = 11). B: 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT, 1.0 mg/kg iv) significantly increased ER and LR by 22 and 85%, respectively (n = 11, P < 0.05 vs. control recordings without drugs). WAY-100635 (1.0 mg/kg iv) significantly decreased ER and LR by 63 and 33%, respectively (n = 8, compared with control recordings without drugs). After 8-OHDPAT, WAY-100635 significantly suppressed ER and LR by 59 and 28%, respectively (n = 11, compared with recordings after 8-OH-DPAT).

Fig. 7

Effects of 8-OH-DPAT on pelvic-EUS reflex when the bladder was distended (0.5 ml of air). In A1 and B1, top traces were obtained without electrical stimulation, and bottom traces were obtained during stimulation. Reflexes were evoked by a single shock to the pelvic nerve. Individual and averaged reflexes were recorded in the same spinal cord-intact animal. A1: spontaneous EUS activity without electrical stimulation before administration of 8-OH-DPAT (top trace) and 2 single sweeps of pelvic-EUS reflex consisting of an ER and some high-amplitude LR bursts (bottom trace). A2: average of 10 individual recordings showing an ER and a few low-amplitude bursts of LR. B1: increase in EUS spontaneous activity without electrical stimulation after administration of 8-OH-DPAT (top trace) and unchanged ER and enhanced LR compared with recordings in A (bottom trace). B2: average of 10 individual recordings showing an ER and variable high-amplitude potentials of LR. Because latencies of LR were shifted and variable after 8-OH-DPAT, LR in an average of 10 individual recordings was difficult to identify. ●, Electrical stimulation (4 V, 1.0 Hz, 0.05-ms pulse width).

Fig. 8

Representative examples of EUS activity and pelvic-EUS reflex after acute spinal cord transection at T8 in an animal with an empty bladder (A) and after distension of the bladder by injection of 0.2 ml of saline (B). Top traces were obtained without electrical stimulation, and bottom traces were obtained during stimulation. Reflexes were evoked by a single shock to the pelvic nerve. After bladder distension, reflexes evoked by the same electrical stimulation were recorded in the same animal. After drug administration, experimental trials were repeated before/after bladder distension. All recordings were obtained within 2–3 h after spinal cord transection. A1: EUS activity in the absence of electrical stimulation when the bladder was empty. A2 and A3: slight enhancement of ER and IR by 8-OH-DPAT (1.0 mg/kg iv) and suppression by WAY-100635 (1.0 mg/kg iv). B1: slight increase in EUS tonic activity in distended bladder. B2: enhancement of EUS tonic activity and LR by 8-OH-DPAT. B3: suppression of tonic EUS activity and LR, but no change in ER, by WAY-100635. Responses represent average of 10 individual reflexes recorded on a computer. ●, Electrical stimulation (6 V, 0.5 Hz, 0.05-ms pulse width).

Fig. 9

Effect of WAY-100635 on facilitation of EUS EMG activity induced by bladder distension. Continuous recording was obtained from 1 spinal cord-intact animal. A: bladder distension by injection of 0.5 ml of air (▲) into the balloon or injection of 1.0 ml of saline (*) into the bladder. At beginning of bladder distension, EUS bursting activity was induced until the bladder was emptied (1). After the bladder was empty, EUS bursts disappeared, leaving only tonic activity (2). Bladder distension (air or saline) was repeated after WAY-100635 (0.1 or 0.4 mg/kg iv). After WAY-100635, duration of EUS bursting and tonic activity was reduced. Balloon was distended until EUS bursting and tonic activity disappeared. B: individual bursts at a faster sweep during time period in A indicated by 1. C: EUS tonic activity at a faster sweep during the time period in A indicated by 2.