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Endocrinology Vol. 147, No. 6 2956-2963
Copyright © 2006 by The Endocrine Society

Estrogen Modulates the Spinal N-Methyl-D-Aspartic Acid-Mediated Pelvic Nerve-to-Urethra Reflex Plasticity in Rats

Sheng-Yen Lin1, Gin-Den Chen1, Jiuan-Miaw Liao, Shwu-Fen Pan, Mei-Jung Chen, Joyce C. Chen, Hsien-Yu Peng, Yu-Cheng Ho, Yen-Chun Ho, Pei-Chen Huang, Jau-Jia Lin and Tzer-Bin Lin

Department of Physiology (S.-Y.L., J.-M.L., H.-Y.P., Y.-Che.H., Y.-Chu.H., T.-B.L.), Graduated Institute of Medical Research (S.-Y.L.), College of Medicine, Department of Obstetrics and Gynecology (G.-D.C.), and Department of Physical Medicine and Rehabilitation (J.-J.L.), Chung-Shan Medical University, Taichung 40201, Taiwan; Department of Biotechnology (S.-F.P.), Ming-Chuan University, Taoyuan 333, Taiwan; Department of Applied Cosmetic (M.-J.C.), Ching-Kuo Institute of Management and Health, Keelong 20346, Taiwan; Department of Anatomy and Cell Biology (J.C.C.), College of Medicine, National Taiwan University, Taipei 106, Taiwan; and School of Medicine (P.-C.H.), Kaohsiung Medical University, Kaohsiung 807, Taiwan

Address all correspondence and requests for reprints to: Dr. Tzer-Bin Lin, Department of Physiology, College of Medicine, Chung-Shan Medical University, No. 110, Chang-Kuo North Road, First Section, Taichung, Taiwan 40201. E-mail: tblin{at}csmu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of gonadal steroids on glutamate-mediated pelvic nerve-to-urethra reflex (PUR) plasticity were investigated in rats, which received a sham operation (Sham), ovariectomy (OVX), or ovariectomy with daily supplemental estrogen (50 µg/kg, OVX + E2). The magnitude of the repetitive stimulation (RS, 1 Hz)-induced potentiation in PUR activity decreased significantly in the OVX group when compared with the Sham groups (18.09 ± 3.91 and 7.40 ± 1.03 spikes/stimulation in Sham and OVX group; respectively, P < 0.01, n = 21). Supplemental estrogen (OVX + E2, 12.60 ± 1.49 spikes/stimulation) significantly reversed the decrease in RS-induced PUR potentiation caused by OVX (P < 0.01, n = 21). The magnitude of the RS-induced potentiation in PUR activity decreased significantly after intrathecal 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline [(20 µM, 10 µl), from 18.09 ± 3.91 to 10.40 ± 0.81, from 7.40 ± 1.03 to 3.20 ± 0.94, and from 12.60 ± 1.49 to 8.06 ± 0.32 spikes/stimulation in Sham, OVX, and OVX + E2, respectively, P < 0.01, n = 18] and D-2-amino-5-phosphonoraleric acid [(100 µM, 10 µl), from 18.09 ± 3.91 to 1.04 ± 0.12, from 7.40 ± 1.03 to 1.06 ± 0.22, and from 12.60 ± 1.49 to 0.98 ± 0.25 spikes/stimulation in Sham, OVX, and OVX + E2, respectively, P < 0.01, n = 18]. In addition, potentiation in PUR activities was induced by intrathecal L-glutamate (0.1 mM, 10 µl, from 1.04 ± 0.02 to 21.60 ± 0.93, from 1.10 ± 0.06 to 8.40 ± 1.50, and from 1.03 ± 0.03 to 18.04 ± 0.84 spikes/stimulation in Sham, OVX, and OVX + E2, respectively, P < 0.01, n = 18) and N-methyl-D-aspartic acid (0.1 mM, 10 µl, from 1.04 ± 0.02 to 14.80 ± 0.97, from 1.10 ± 0.06 to 4.60 ± 0.48, and from 1.03 ± 0.03 to 9.09 ± 0.63 spikes/stimulation in Sham, OVX, and OVX + E2); N-methyl-D-aspartic acid-mediated PUR plasticity in female rats and may contribute to alterations in urinary dysfunction after menopause.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MICTURITION IS A complex function that needs the coordination of the sympathetic, parasympathetic, and somatic nervous systems. During the storage phase of a micturition cycle, impulses induced by bladder distension transmit centripetally onto the dorsal horn neurons through the pelvic afferent fibers. After integrating within the spinal cord, these impulses cause external urethral sphincter contraction via the pudendal efferent nerve. This pelvic nerve-to-urethra reflex (PUR) is essential for urine continence (1).

The reflex plasticity means the efficacies of a reflex can vary, depending on the neuronal circuitry within the nervous system as well as the patterns of ongoing activities (2). Several types of reflex plasticity have been widely investigated for their presumed relationship to memory (3, 4) and hypergasia (5, 6). Long-term potentiation is characterized by a long-lasting enhancement in efficacies of excitatory synapses after a strong repetitive stimulation of input fibers (7, 8, 9). Another activity-dependent reflex plasticity, termed windup, is a progressive increase in the spike number evoked per stimulus that occurs in dorsal horn neurons of the spinal cord under a low-frequency repetitive stimulation (10). Recent studies on reflex plasticity, using intact spinal cord preparations, have demonstrated an N-methyl-D-aspartate (NMDA)-mediated plasticity in PUR induced by repetitive (11, 12) and tetanic (13) peripheral inputs, indicating an important role for spinal NMDA-dependent reflex plasticity in micturition functions.

Estrogen is a gonadal steroid with pronounced tropic effects on many diverse populations of neurons throughout the peripheral and central nervous systems. Both clinical and experimental evidences suggest that estrogen has a neurotherapeutic role after neurological injury or disease, including the enhancement of regenerative properties of injured motor neurons (14, 15). Intracellular recordings have shown that estradiol (E2) administration increases the synaptic excitation of pyramidal cells in CA1 area, suggesting that gonadal steroid levels impact reflex plasticity. Studies show that pharmacological or surgical ablation of menses (or castration) decrease the duration of the excitatory postsynaptic potential in the hippocampus (16, 17, 18). Some experiments have also shown that the synaptic plasticity in the CA1 area is modulated by the menstrual cycle (3, 16) or hormone replacement therapy (20). A more recent study demonstrated that E2-treated rats had an increased level of NMDAR1 mRNA and protein in the pyramidal neurons of the CA1 area. These findings explain the greater sensitivity of these neurons to NMDA- but not to AMPA-mediated synaptic inputs in E2-treated rats (20). The NMDA-mediated synaptic inputs are not only in the CA1 area of the hippocampus but also shown in the spinal cord. The glutamatergic-NMDA receptor is widely used for primary afferent neurotransmission at the spinal levels controlling micturition functions (21).

In this study, to examine the role of gonadal hormones on the PUR plasticity, we tested the effects of ovariectomy and estrogen replacement on stimulation-induced responses in anesthetized rats. In addition, glutamatergic agonists/antagonists were used to elucidate the possible neurotransmitter(s) involved in the stimulation-induced PUR plasticity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal preparations
Sixty-three adult female Sprague Dawley rats (210–275 g) were used in this experiment. Animals were randomly divided into three groups (21 rats in each group). One group of rats was ovariectomized (OVX) and tested 20–30 d after the surgery. Another group was ovariectomized and given daily sc injections of 50 µg/kg 17-ß-estradiol-3 benzoate dissolved in 100 ml sesame oil (OVX + E2) at least 20 d after the ovariectomy (mean = 22.5 ± 0.76 d). Sham operations (Sham) were performed on the other group to serve as the control. On the days of experiments, rats were anesthetized with urethane (1.2 g/kg, ip). The animal care and experimental protocol were in accordance with the guidelines of The National Science Council in Taiwan. All efforts were made to minimize both animal suffering and the number of animals used throughout the experiment. The trachea was intubated to keep the airway patent. A PE-50 catheter (Portex, Hythe, Kent, UK) was placed in the left femoral vein for administration of anesthetics when needed. The body temperature was kept at 36.5–37.0 C by an infrared light and was monitored using a rectal thermometer. The rats were monitored for the corneal reflex and a response to noxious stimulation to the paw throughout the experiment. If either was present, a supplementary dose (0.4 g/kg, iv) of anesthetics was given through the venous catheter. At the end of the experiments, the animals were killed under deep anesthesia, using an iv injection of potassium chloride saturation solution.

Surgical preparations
A midline abdominal incision was made to expose the pelvic viscera. Both ureters were ligated caudally and cut rostrally at the ligated sites. To avoid any fluid retention in ureters, the proximal ends of the ureters drained freely within the abdominal cavity. A wide-bore cannula, with a sidearm for the pressure measurement, was tied into the lumen of the bladder at the apex of the bladder dome. To keep urinary bladder empty, the urine was drained freely through the bladder cannula. In some experiments recording the intraurethral pressure (IUP), two 4-0 silk sutures were placed around the bladder trigone and ligated. A wide-bore urethra cannula was inserted through the opening of the urethra and connected to a pressure transducer and continuously recorded on the computer system (MP30, Biopac, Santa Barbara, CA).

Intrathecal catheter
The occipital crest of the skull was exposed and the atlantooccipital membrane was incised at the midline with the tip of an 18-gauge needle. A PE-10 catheter was inserted through the slit and passed caudally to the L6 level of the spinal cord. The volume of fluid within the cannula was kept constant a 10 µl in all experiments. Single 10-µl volumes of drug solutions were administered followed by a 10-µl flush of artificial cerebrospinal fluid (22). The length of time of the injection was about 2–4 sec. At the end of the experiment, a laminectomy was performed to verify the location of the cannula tip.

Nerve dissection and stimulation
The right pelvic nerve was dissected carefully from the surrounding tissue and then transected as distal as possible. The stimulated nerve and the electrodes were bathed in a pool of warm paraffin oil (37 C) to prevent drying. An electric current of square wave pulses with pulse durations of 0.1 ms was applied from a stimulator (Grass S88, Cleveland, OH) through a stimulus isolation unit (Grass SIU5B) with a constant current unit (Grass CCU1A).

Recording of electromyogram activity
Epoxy-coated copper wire (50 µm; M. T. Giken Co., Tokyo, Japan) electromyogram electrodes were placed in the skeletal external urinary sphincter. This was performed using a 30-gauge needle with hooked electromyogram electrodes positioned at the tip. The needles were inserted into the sphincter approximately 1–2 mm lateral to the urethra and then withdrawn, leaving the electromyogram wires embedded in the muscle. The external urethral sphincter electromyogram (EUSE) activities were amplified 20,000-fold and filtered (high frequency cut-off at 3000 Hz and low at 30 Hz, respectively) by a preamplifier (Grass P511AC) and then continuously displayed on an oscilloscope (Tectronics TDS 3014, Wilsonville, OR) and the recording system (MP30, Biopac). The stimulated nerve and the electrodes were bathed in a pool of warm paraffin oil (37 C) to prevent drying.

Experimental arrangement
The schematic arrangement of EUSE recordings, as well as the pelvic afferent nerve fiber stimulation, are shown in Fig. 1AGo. The protocol for assessing the PUR activities was as follows. Once the electrodes’ positions were optimized, recording of EUSE activities began. Single shocks were repeated at 30-sec intervals [referred to as test activity (TS)] and given through a pair of stimulation electrodes. This frequency of stimulation was chosen for sampling data because it did not result in response facilitation. The intensity of stimulation was gradually increased from 0 to 30 V and a stimulus intensity that yielded a single spike action potential in the EUSE was usually chosen to standardize the baseline reflex activity. After the baseline period (30 min), a repetitive stimulation, at 1 Hz that lasted for 30 min with intensity identical with the test stimulation [referred to as repetitive activity (RS)], was applied to induce facilitation in reflex.


Figure 1
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  		FIG. 1. The pelvic nerve-to-urethra reflex activities. A, A schematic arrangement of the EUSE activities and IUP recording and the pelvic afferent nerve stimulation (Stim). B, An action potential in EUSE evoked by a single electric shock (indicated by a triangle at the bottom) at the pelvic afferent nerve in rats that received a sham operation (Sham), OVX, or OVX + E2. C, The latencies of the pelvic nerve-to-urethra reflex in the three groups. D, The numbers of action potentials in EUSE evoked by the TS (1/30 Hz, for 30 min) in rats that received a sham operation (circle), OVX (square), and OVX + E2 (triangle). Abscissa, The spike numbers evoked by TS (P > 0.05, n = 21).

 
Application of drugs
Drugs were administered by intrathecal injection through the intrathecal catheter with a solution of known drug concentrations. To investigate the possible neurotransmission involved in this study, glutamatergic NMDA receptor selective antagonist, D-2-amino-5-phosphonoraleric acid (APV; 100 µM, 10 µl, Sigma, St. Louis, MO) and {alpha}-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) receptor selective antagonist, 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline (NBQX; 20 µM, 10 µl, Sigma) were tested intrathecally (injection rate was about 5 µl/sec). In addition, two agonists, glutamate and NMDA, were also used to test through the same route. Saline of identical volume to tested agents was dispensed intrathecally to serve as a vehicle. Each tested agent was injected 3 min before repetitive stimulation started. Experimental animals received all the five tested agents (saline included). A flush of saline (20 µl) was injected 1 min after the end of each test and was followed by an equilibrium period (30 min) before the next test. At the end of the experiment, the location of the injection site was marked by an injection of Alcian blue (10 µl, 2%). The volume of drug injected into the spinal cord in this experiment has been reported to spread from 0.5 to 1.5 mm from the site of the injection (22). Therefore, a cannula positioned more than 0.5 mm from the intended site of injection was not included in the statistical analysis. There were two to three animals in each group, in which the tip of the intrathecal catheter deviated by more than 0.5 mm from the target structure (i.e. the entry zone of L5-S1 spinal segments). These animals were excluded from the analysis.

Data analysis
The spike numbers from the external urethral sphincter electromyogram were counted every 10 sec and averaged. The effects of test agents were evaluated by the percentage of changes in spike numbers caused by the tested agents [i.e. (spike numbers before treatment – spike numbers after treatment) x 100/spike numbers before treatment] percent. All the data in the text and figures are mean value ± SEM. Statistical analysis of the data were performed by means of ANOVA. P < 0.05 was accepted as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The PUR activities
Activities recorded from the EUSE, elicited by a single electric shock of the pelvic afferent nerve obtained from rats that had received a Sham, OVX, or OVX + E2 are shown in Fig. 1BGo. The mean reflex times for the pelvic nerve afferent fiber stimulations to evoke an action potential in the EUSE were 52.50 ± 4.73, 57.67 ± 2.11, and 55.00 ± 3.98 ms in the Sham, OVX, and OVX + E2 groups, respectively (n = 21). No statistical differences were found in the latencies of stimulation-induced PUR between either two of these three groups (Fig. 1CGo, P > 0.05, n = 21). The numbers of action potentials in EUSE activities elicited by the test stimulation (1/30 Hz for 30 min) in Sham, OVX, and OVX + E2 are shown in Fig. 1DGo. No statistical significance was found in the spike numbers evoked by the test stimulation between either two of these three groups within the test period.

Changes in PUR activities induced by repetitive stimulation
The excitability of PUR was assessed by recording action potentials of EUSE activities resulting from stimulations of the pelvic afferent nerve with electrical shocks of single pulses derived at a frequency of 1/30 Hz (TS). As shown in Fig. 1DGo, the EUSE activities varied little over the 30-min testing period. However, in these groups, including Sham, OVX, and OVX + E2, potentiations in PUR activities were induced progressively when an RS (at the same intensity as the test stimulation, delivered at 1 Hz for 30 min) was applied to the pelvic afferent nerve (Fig. 2AGo). The potentiations in PUR activities induced by the repetitive pelvic nerve stimulation in these three groups are summarized in Fig. 2BGo. The RS-induced potentiation was significantly lower in the OVX when compared with the Sham group (P < 0.05, n = 21), whereas no statistical difference was found between the Sham and OVX + E2 groups (P > 0.05, n = 21).


Figure 2
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  		FIG. 2. Potentiation in pelvic nerve-to-urethra reflex activities caused by repetitive stimulation. A, Single action potentials in EUSE were evoked by pelvic afferent nerve TS (1/30 Hz, indicated by stimulation artifacts at the lower tracing). A potentiation in pelvic nerve-to-urethra reflex activities was caused by RS (1 Hz for 30 min, indicated by stimulation artifacts at the lower tracing) at the pelvic afferent nerve in rats that received Sham, OVX, and OVX + E2. The tracing before break symbol (left) shows the EUSE activities evoked by the repetitive stimulation during the first 7 sec after stimulation onset. The tracing after the break symbol (right) shows the EUSE activities during the last 7 sec of the stimulation period (30 min). B, Summarized data (mean ± SEM) showing the potentiation in pelvic nerve-to-urethra reflex activities induced by the RS (1 Hz, for 30 min) in rats that received Sham (square), OVX (triangle), OVX + E2 (reversed triangle), and that done by TS (1/30 Hz for 30 min). Abscissa, The spike numbers evoked by the RS. *, P < 0.05, **, P < 0.01, significant difference from TS; #, P < 0.05 significant difference from Sham group; +, P < 0.05 significant difference from OVX group (n = 21).

 
Secondary changes in response to plasticity
As shown in Fig. 3AGo, in the Sham group, the firing in EUSE increased when the pelvic afferent fiber was stimulated using the RS (1 Hz). Furthermore, the duration of the contraction wave of IUP, secondary to the urethral sphincter contraction, increased. In the OVX group, RS also elicited a potentiation of PUR activities with a lower firing frequency and the duration of the contraction wave in IUP was also shorter than in the Sham group (Fig. 3BGo, OVX). In addition, a daily supplement of estrogen reversed the effects of OVX on PUR and IUP activities (Fig. 3CGo, OVX + E2).


Figure 3
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  		FIG. 3. Changes in IUP resulting from the RS (1 Hz)-induced potentiation in pelvic nerve-to-urethra reflex activities. A, The potentiated firings in the EUSE caused successive contraction waves in IUP in rats that received Sham. B, Repetitive stimulation-induced potentiation in EUSE activities and the numbers of contraction waves in IUP was reduced in rats that received OVX. C, A daily supplement of estrogen (OVX + E2) reversed the effects of OVX on PUR and IVP activities.

 
Antagonism by NBQX and APV
As shown in Fig. 4AGo, intrathecal NBQX (RS + NBQX), the novel selective non-NMDA receptor antagonist, caused a reversible antagonism on the RS-induced potentiation in PUR activities in all three groups (P < 0.01, n = 18). In addition, there was no statistical difference shown in the antagonistic effects caused by NBQX in either two of the three groups (P < 0.05, n = 18). On the other hand, APV (RS + APV), the selective NMDA receptor antagonist, abolished the repetitive stimulation-induced potentiation in PUR activities in these three groups (P < 0.01, n = 18). In addition, the antagonistic effect caused by APV was significantly decreased in OVX group when compared with Sham group (P < 0.01, n = 18). Furthermore, supplemental estrogen significantly reversed the antagonistic effect caused by APV (OVX + E2; P < 0.05, n = 18). Saline of identical volume was also tested through the same route but showed no effects on the repetitive stimulation-induced potentiations. In six animals (two in each group), NBQX and APV were injected before the TS (1/30 Hz) but showed no effect on the TS-induced EUSE activity. Figure 4BGo summarizes the effects of NBQX and APV on the RS-induced potentiation in PUR activities in these three groups.


Figure 4
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  		FIG. 4. The antagonism of NBQX and APV on RS (1 Hz)-induced potentiation in pelvic nerve-to-urethra reflex activities. A, Column 1, An action potential in EUSE activities was evoked by the TS 1(/30 Hz, indicated by the stimulation artifact at the lower tracing) at the pelvic afferent nerve in rats that received Sham, OVX, and OVX + E2. Column 2, RS induced a potentiation in pelvic nerve-to-urethra reflex activities in the three groups. Column 3, NBQX attenuated RS-induced potentiation in pelvic nerve-to-urethra reflex activities in the three groups. Column 4, APV abolished RS-induced potentiation in pelvic nerve-to-urethra reflex activities in the three groups. B, Summarized data (mean ± SEM) showing the effects of antagonism of NBQX and APV on the repetitive stimulation-induced potentiation in pelvic nerve-to-urethra reflex activities. Abscissa, The spike numbers evoked by repetitive stimulation. **, P < 0.01, significant difference from Sham group; ##, significant difference from OVX group, n = 18).

 
Agonist-induced potentiations in PUR activities
As shown in Fig. 5AGo, intrathecal L-glutamate (TS + Glu) caused a potentiation in PUR activities induced by the TS (1/30 Hz) in these three groups (P < 0.01, n = 18). NMDA (TS + NMDA) also elicited a potentiation in PUR activities evoked by the test stimulation in the same preparations (P < 0.01, n = 18). The potentiation in PUR activities caused by intrathecal glutamate and NMDA were decreased in OVX group when compared with the Sham group (both P < 0.01, n = 18). Furthermore, supplemental estrogen (OVX + E2) significantly reversed the decrement in agonists-induced potentiation when compared with the OVX group (both P < 0.01, n = 18). Figure 5BGo summarizes the effects of intrathecal glutamate and NMDA on testing stimulation-induced PUR activities in these three groups.


Figure 5
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  		FIG. 5. The agonist-induced potentiation in pelvic nerve-to-urethra reflex activities. A, Column 1, An action potential in EUSE activities was evoked by the TS (1/30 Hz, indicated by the stimulation artifact at the lower trace) at pelvic afferent nerve in rats that received Sham, OVX, and OVX + E2. Column 2, Glutamate (TS + Glu) potentiated the external urethral sphincter activities evoked by the TS in the three groups. Column 3, Glutamatergic NMDA (TS + NMDA) potentiated the pelvic nerve-to-urethra reflex activities in the three groups. B, Summarized data (mean ± SEM) showing the effects of glutamate and NMDA on the pelvic nerve-to-urethra reflex activities evoked by test stimulation. Abscissa, The spike numbers evoked by repetitive stimulation. **, P < 0.01, significant difference from Sham group; ##, significant difference from OVX group, n = 18).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The tissue of the lower urinary tract and pelvic floor are known to be estrogen selective because estrogen receptors are located throughout the bladder and urethra (23, 24). Cystometrical investigations on animals have shown a decrease in maximal voiding pressure (25) and voiding volume (26) and an increase in threshold and intervoiding pressure (27) after OVX. During the storage phase of a voiding cycle, urine distension excites the mechanical receptors on the bladder wall and generates action potentials transmitting centripetally into the spinal cord through the pelvic afferent fiber. These impulses, after integrating within the spinal level, in turn, excite the urethral sphincter via the pudendal efferent nerve and produce continence (1). Not only the PUR itself but also the stimulation-induced reflex plasticity on this reflex are interesting topics for their presumed relationship to urinary continence (11, 12, 13).

The results from the present in vivo electrophysiology experiments demonstrate that without affecting the baseline activities, estrogen modulates the neural plasticity of the PUR. In addition, the physiological functions secondary to the plasticity, i.e. the contraction wave of the urethra, are also affected. These results provide possible mechanisms for the urodynamic changes observed in clinical observations, where the symptoms of stress urinary incontinence resulting from insufficient urethral resistance do not often appear until menopause and results suggest that gonadal hormones are important for the modulation of some aspects of micturition functions.

It has been known for some time that neural plasticity depends on the availability of glutamatergic NMDA receptors (21 , and coincidentally, estrogen increases the numbers of NMDA-type binding sites on the hippocampal CA1 neuron (13). Woolley et al. (20) recently confirmed this finding and proposed that this increase affinity may be explained by the greater concentration of NMDAR1 receptor protein, which is regulated by the level of estrogen. These changes in NMDA binding may, in turn, result in greater sensitivity to NMDA-dependent synaptic inputs (18). Glutamatergic NMDA receptors are widely used in the spinal cord level for primary afferent neural transmission (28). In addition, recent studies using an intact spinal cord preparation have demonstrated a NMDA-dependent PUR plasticity in the spinal cord level (11, 12, 13). In the present study, intrathecal glutamatergic antagonists blocked the estrogen-related PUR plasticity, providing a pharmacological basis for estrogen modulation on glutamate-mediated reflex plasticity at the spinal cord level.

In the present study, glutamatergic AMPA and NMDA receptor antagonists reduced RS-induced reflex plasticity. In addition, E2 replacement reversed the antagonistic effect elicited by NMDA but not AMPA receptor antagonist. This result indicated the deficit in RS-induced PUR plasticity caused by estrogen depletion in this study was mainly resulted from glutamatergic NMDA receptor-dependent mechanism. This conjecture was further supported by the results that E2 supplement can significantly reverse the decrement in NMDA-induced reflex potentiation in OVX group.

Lumbosacral dorsal root ganglion neurons are immunoreactive for estrogen receptor-{alpha}/ß (28, 29, 30, 31). Immunohistochemistry studies, investigating the spinal cord, have shown that estrogen receptors are expressed in dorsal horn neurons (32, 33). Therefore, the simplest explanation for the present results would be that treatment with estrogen increases the availability of NMDA receptors, providing the grounds for facilitated potentiation of synaptic strength.

Wong and Moss (16) found that E2 priming, 2 d before obtaining the hippocampal slices, increased synaptic excitability by prolonging excitatory postsynaptic potential and inducing repetitive firing in response to Schaffer collateral stimulation in a small percentage of CA1 neurons recorded in vitro with intracellular electrodes. However, in a similar preparation, Wooley et al. (20) found no difference in the efficacy of synaptic input, unless the postsynaptic response had been stripped of AMPA-dependent components. In comparison with these results, Warren et al. (34) did not observe changes in basic excitability measured by I/O curves during the estrous cycle of chronically implanted rats. In the present experiments, baseline excitability remained unmodified after an ovariectomy as evidenced by a similar latent reflex in ovariectomized with Sham rats. Therefore, we may conclude the levels of E2, which may reduce the sensitivity to NMDA-mediated glutamate input, did not affect the baseline response of PUR.

Not only does estrogen affect the neural pathways within the central nervous system, there are also studies investigating the efferent nerve innervating the urethra have indicated that the neurotransmitter released from the motor nerve decreased significantly after estrogen depletion (35). In addition, investigations also indicated that the tensions resulted from spontaneous (36) and electric shock-induced (19) contractions in urethra musculature decreased significantly after ovariectomy, i.e. estrogen may directly affect the urethra muscle. Whether this may be a confounding factor in this study need be further investigated to clarify the underlying mechanism involved in the changes in PUR reflex activity demonstrated in this study.

In conclusion, the present electrophysiological studies clearly demonstrate that estrogen has a significant role in modulating urethra activity. The loss of gonadal hormones decreases sensitivity and estrogen replacement reverses the impairment in urethra functions. The decrease in the NMDA-mediated RS-induced PUR plasticity may implicate a decrease in processing in the spinal cord micturition circuitry. Our data further suggest that alterations in neural processing within the spinal cord, mediated by estrogen, may underlie symptoms of stress urinary incontinence.


   Footnotes
 
This work was supported by The National Science Council (NSC) in Taiwan (NSC 93-2320-B-040-065 and 94-2320-B-040-026 to T.-B.L. as well as NSC 93-2314-B-040-013 and 94-2314-B-040-026 to G.-D.C.).

First Published Online March 16, 2006

1 S.-Y.L. and G.-D.C. contributed equally to this study. Back

Abbreviations: APV, D-2-Amino-5-phosphonoraleric acid; AMPA, {alpha}-amino-3-hydroxy-5-methylisoxazole-propionic acid; E2, estradiol; EUSE, external urethral sphincter electromyogram; IUP, intraurethral pressure; NBQX, 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline; NMDA, N-methyl-D-aspartate; OVX, ovariectomy; OVX + E2, OVX with daily supplemental estrogen; PUR, pelvic nerve-to-urethra reflex; RS, repetitive stimulation activity; Sham, sham operation; TS, test stimulation activity.

Received October 31, 2005.

Accepted for publication March 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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