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Am J Physiol Renal Physiol 293: F1115-F1122, 2007. First published July 18, 2007; doi:10.1152/ajprenal.00135.2007
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Descending facilitation of spinal NMDA-dependent reflex potentiation from pontine tegmentum in rats

Gin-Den Chen,1 Hsien-Yu Peng,2,3 Kwong-Chung Tung ,2,* Chen-Li Cheng,4 Yi-Jui Chen,5,6 Jiuan-Miaw Liao,3 Yu-Cheng Ho,3 Shwu-Fen Pan,7 Mei-Jung Chen,8 and Tzer-Bin Lin3,9,*

1Department of Obstetrics and Gynecology, Chung-Shan Medical University Hospital, 3Department of Physiology, College of Medicine, Chung-Shan Medical University, 2Department of Veterinary Medicine, National Chung-Hsing University, and 4Division of Urology, Department of Surgery, Taichung Veterans General Hospital, Taichung; 5Department of Nursing, Cardinal Tien College of Healthcare and Management, Taipei; and Departments of 6Pharmacy and 9Medicine, St. Paul's Hospital, and Departments of 7Biotechnology and 8Biomedical Engineering, Ming-Chuan University, Taoyuan, Taiwan

Submitted 22 March 2007 ; accepted in final form 16 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was conducted to investigate whether dorsolateral pontine tegmentum stimulation modulates spinal reflex potentiation (SRP) and whether serotonergic neurotransmission is involved in such a modulation. Reflex activities of the external urethra sphincter (EUS) electromyogram in response to a test stimulation (TS; 1/30 Hz) or repetitive stimulation (RS; 1 Hz) on the pelvic afferent nerve in 35 anesthetized rats were recorded with/without synchronized train pontine stimulation (PS; 300 Hz, 30 ms) and/or intrathecal administrations of 10 µl of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline (NBQX; 100 µM), D-2-amino-5-phosphonovalerate (APV; 100 µM), N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride (WAY 100635; 100 µM), and 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT; 100 µM). The TS evoked a single action potential (1.00 ± 0.00 spikes/stimulation), while the RS produced a long-lasting SRP (16.12 ± 1.59 spikes/stimulation) that was abolished by APV (1.57 ± 0.29 spikes/stimulation) and was attenuated by NBQX (7.42 ± 0.57 spikes/stimulation). Synchronized train PS with RS (PS+RS) produced facilitation in RS-induced SRP (25.17 ± 2.21 spikes/stimulation). Intrathecal WAY 100635 abolished the facilitation in SRP as a result of the synchronized PS (14.66 ± 1.58 spikes/stimulation). On the other hand, intrathecal 8-OH-DPAT elicited facilitation in the RS-induced SRP (25.16 ± 1.05 spikes/stimulation) without synchronized PS. Our findings suggest that dorsolateral pontine tegmentum may modulate N-methyl-D-aspartic acid-dependent SRP via descending serotonergic neurotransmission. This descending modulation may have physiological/pharmacological relevance in the neural controls of urethral closure.

long term potentiation; SRP; pontine tegmentum; serotonin; WAY 100635; 8-OH-DPAT

ACTIVITY-DEPENDENT REFLEX plasticity, known as long-term potentiation (4, 5, 42) and windup phenomenon (37, 50, 51, 58), has been widely studied in the central neural system, including the hippocampus and the spinal cord, for exploring the mechanisms of memory (3, 41) and hypergesia (46, 57), respectively. Forms of activity-dependent reflex plasticity can be elicited by applying electric shocks (6, 13) and by reagent injections/perfusions to specific sites (24). On the other hand, an established/establishing reflex plasticity may be modified by genetic (50), pharmacological (23, 46), surgical (27, 48, 54), or behavioral manipulations (2, 47). To the best of our knowledge, few studies have explored the possibility of modulating activity-dependent reflex plasticity by activating the higher nucleus of a specific neural projection to the site, where the reflex plasticity occurs. However, modulating activity-dependent reflex plasticity from a neural nucleus may mimic the physiological conditions of neural functions and offer gateways to elucidate the physiological/pharmacological relevancies in such activity-dependent reflex plasticity (1).

Kuru (29) emphasized the importance of understanding the descending innervations from the brain stem to modulate the spinal reflexes involved in pelvic viscera, including the urinary bladder. Several researchers have applied electric shocks on the pontine reticular formation, which is the regulation center of micturition functions, to modulate the electric activity coming from lumbosacral spinal neurons to innervate the urinary bladder (22, 44). These findings reveal an important role of the pontine reticular formation in descending control on spinal integrated micturition functions.

Serotonin (5-hydroxytryptamine; 5-HT) has been shown to play an important role in the descending control of micturition functions (7, 8, 15, 16, 30, 55). Serotonin receptors are widely distributed in the lumbosacral spinal cord, which is the origin of parasympathetic outflow (20, 43, 44, 56). However, the real role of 5-HT in descending neural control has been difficult to evaluate because of the existence of multiple subtypes of 5-HT receptors and the lack of receptor subtype-specific agonists/antagonists (33, 39). Among 5-HT-receptor subtypes, the 5-HT1A receptor has been well investigated due to the early availability of a selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) (17, 19). Previous experiments have revealed that the activation of supraspinal 5-HT1A systems modulates the spinal integrated reflex in rats (30) and show the importance of the 5-HT1A receptors in the descending control of the spinal reflex activities.

Recently, using in vivo animal preparations, we demonstrated that low-frequency repetitive stimulations (RS) on the pelvic afferent nerve might elicit a novel form of activity-dependent reflex plasticity, i.e., a spinal reflex potentiation (SRP) in pelvic nerve-to-EUS reflex activity (9, 10, 31–36). Such an animal model is quite different from investigations using brain or spinal slices with a thickness of several millimeters and may maintain the whole neural network within the central nervous system as well as all the dorsal and ventral rootlets attached on the spinal cord intact, thereby offering a gateway to investigate modulations on the SRP resulting from a specific neuronal projection to the site where SRP occurs. In the present study, we investigated whether the pontine descending innervations modify SRP in vivo animal preparations. We also tested whether an intrathecal 5-HT1A agonist/antagonist, at the spinal level, mediates such a descending modulation coming from the pons varolli.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
General Preparations

Thirty-five adult female Wistar rats, weighing 250–350 g, were anesthetized with urethane (1.2 g/kg ip). Animal care and the experimental protocol were approved by the National Science Council in Taiwan. 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. Body temperature was kept at 36.5–37.0°C by infrared light and was monitored using a rectal thermometer. The corneal reflexes of the rats and responses to a noxious stimulation to the paw were monitored throughout the experiment. If responses were present, a supplementary dose (0.4 g/kg) of urethane was given through the venous catheter. After all the experiments were completed, the animals were euthanized via an intravenous injection of potassium chloride saturation solution.

Intrathecal Catheterization

The occipital crest of the skull was exposed, and the atlantooccipital membrane was incised at the midline with the tip of a 24-gauge needle. A PE-10 catheter was inserted through the slit and passed caudally to the L6 level of the spinal cord, the level presumed to mediate the micturition reflex. Test reagents were injected 1 min before the test stimulation (TS)/RS onset. The volume of fluid within the cannula was kept constant at 10 µl in all experiments. Single 10-µl volumes of drug solutions were administered followed by a 10-µl flush of artificial cerebrospinal fluid. At the end of the experiments, the location of the injection site was marked by an injection of Alcian blue (10 µl, 2%), followed by a laminectomy, which was performed to verify the location of the cannula tip. The volume of drug injected into the spinal cord in this experiment may spread from 0.5–1.5 mm from the site of injection as previously described (41). The experimental animals, in which the cannula tip deviated by >0.5 mm from the target structure were excluded from statistical analysis.

Dorsolateral Pontine Tegmentum Stimulation

The dorsolateral pontine tegmentum was stimulated to activate the descending limb to the spinal reflex circuitry. This procedure was done as follows. The rat was placed in a stereotaxic apparatus, and a small craniotomy was performed to insert an electrode into the dorsal pontine tegmentum. Fine concentric bipolar stainless steel wire electrodes (diameter 0.1–0.2 mm, Giken, Tokyo, Japan), insulated with Teflon except for the tip, were used for stimulation in the brain stem.

A transurethral bladder cannula was inserted into the urinary bladder from the urethra opening. A ligation around the urethra opening was done to immobilize the transurethral cannula and avoid fluid leakage from the bladder through the urethra. This cannula was connected to a pressure transducer (P23 ID, Gould-Statham, El Segundo, CA) which was connected to a computer system (MP30, Biopac, Santa Barbara, CA) and through a preamplifier (Grass 7P1, Cleveland, OH) for recording intravesicular pressure (IVP). Saline was injected through the transurethral bladder cannula to determine the threshold volume for inducing rhythmic isovolumic contractions. After the threshold volume was obtained, small amounts of saline were withdrawn from the bladder until bladder contractions disappeared. The bladder volume was kept isovolumically below the threshold for inducing spontaneous bladder contractions during brain stem stimulation. The electrodes were then introduced stereotaxically into the lateral part of the dorsal pontine tegmentum (AP, –8.4 to –9.4 mm; L, 1.0 to 1.5 mm; H, –6.5 to –7.0 mm from the bregma) in 0.25- or 0.5-mm steps using a micromanipulator (25). Electrical stimulation in the dorsolateral pontine tegmentum consisted of sequences of stimulation (1–15 V, 0.05 ms in pulse duration at 300 Hz, 5-ms train duration). The optimal sites for inducing an isovolumic bladder contraction in the bladder, with the largest amplitude (usually >15 mmH2O), were determined in each experiment. After localization of the stimulation point at the pons varolli, the ligation around the urethra opening was cut and the transurethral bladder cannula was withdrawn. The lower body of the rat was rotated to a supine position, and a midline abdominal incision was made to expose the pelvic viscera. Both ureters were ligated distally and cut proximally to the ligation sites. The proximal ends of the ureters drained freely into the abdominal cavity. Another wide-bore intravesical bladder cannula was tied into the apex of the bladder dome. The urinary bladder was drained free to rule out unnecessary afferent inputs. Abdominal skin flaps were tied to a metal frame to form a skin pool, and the abdominal cavity was filled with warm mineral oil. After all the experimental procedures were finished, the stimulation site was electrically injured by passing a DC current through it, and then the site was reconstructed with reference to the lesion mark. Experimental animals in which the stimulation site deviated by >1 mm from the target structure were excluded from statistical analysis.

Nerve Dissection and Stimulation

The left pelvic nerve was dissected carefully from the surrounding tissue and was transected distally, while the right pelvic nerve was left intact. The dissected nerve was placed on a pair of stainless steel wire electrodes and stimulated by an electric current of square-wave pulses with pulse durations of 0.1 ms applied by a stimulator (Grass S88) through a stimulus isolation unit (Grass SIU5B) and a constant-current unit (Grass CCU1A).

Reflex Activity Recording

Epoxy-coated copper wire (50 µm, Giken) electromyogram electrodes were placed into the periurethra area intra-abdominally. This procedure was performed using a 30-gauge needle with a hooked electromyogram electrode positioned at the tip (1.0–1.5 mm). The needle was inserted into the sphincter ~1–2 mm lateral to the urethra and then withdrawn, leaving the electromyogram wire embedded in the sphincter. The electromyogram activities were amplified 20,000-fold and filtered (high-frequency cut-off at 3,000 Hz and low at 30 Hz, respectively) by a preamplifier (Grass P511AC), then continuously displayed on an oscilloscope (Tectronics TDS 3014, Wilsonville, OR) and the recording system with a sampling rate of 20,000 Hz (MP30, Biopac).

Experimental Arrangement and Protocol

The schematic arrangement of reflex activity recordings, in response to the afferent nerve stimulation with/without pons varolli stimulation, is shown in Fig. 1A. After the urinary bladder drained free via the intravesical bladder cannula, the experimental protocol was as follows.


Figure 1
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  		Fig. 1. Effects of pelvic afferent nerve stimulation and train stimulations on the pons varolli. A: schematic arrangement of external urethra sphincter electromyogram (EUSE) recording in response to pelvic afferent nerve and/or pontine stimulations (Stim). VC, visual cortex; IC, inferior colliculi; PAG, periaqueductal gray; Py, pyramidal tract. Dots indicate locations of the tips of the stimulating electrodes. B: short-train pulse pontine stimulation (PS) produced an increase in intravesicular pressure (IVP) in a saline-filled urinary bladder. C: after the urinary bladder was drained free, a short-train pulse PS evoked no action potential in electromyogram activities. D: when the urinary bladder was drained free, a single action potential in the reflex activities was induced by pelvic afferent nerve test stimulation (TS).

 
Protocol I: TS. Single shocks at fixed suprathreshold strengths (5–30 V) were repeated at 30-s intervals and were 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 reflex activities was usually chosen to standardize the baseline reflex activity.

Protocol II: RS. After a baseline period (usually 30 min), RS (1 Hz, lasting for 30 min) with an intensity identical to the TS was applied to induce SRP.

Protocol III: glutamatergic agonist/antagonist. After a stimulation-induced SRP had been established by repetitive pelvic afferent nerve stimulation, glutamatergic N-methyl-D-aspartic acid (NMDA; 100 µM, 10 µl) or an AMPA receptor antagonist, i.e., D-2-amino-5-phosphonovalerate (APV) or 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline (NBQX), was tested intrathecally. After flushing and an equilibrium period (usually 30 min), TS was used to evoke the baseline reflex activity once again. A glutamatergic agonist including glutamate (100 µM, 10 µl) and NMDA (100 µM, 10 µl) were then tested intrathecally to elucidate the possible neurotransmitters involved in SRP.

Protocol IV: synchronized train pontine stimulation with RS. Pontine train stimulation (synchronized with afferent fiber stimulation, i.e., 1 Hz in train rate, 300 Hz in pulse frequency, 5-ms train duration) accompanied by the afferent nerve RS (1 Hz, lasting for 30 min; PS+RS) with an intensity identical to protocol II was applied to induce facilitation in SRP.

Protocol V: antagonist administration. A selective 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N(2-pyridinyl) cyclohexanecarboxamide trihydrochloride (WAY 100635, 100 µM, 10 µl), was administered intrathecally 2 min before the synchronized train PS with the RS (with identical parameters to protocol IV).

Protocol VI: agonist administration. A selective 5-HT1A receptor agonist, 8-OH-DPAT (100 µM, 10 µl), was administered intrathecally 2 min before the afferent nerve RS (with identical parameters to protocol II).

Application of Drugs

Drugs were administered by intrathecal injection, including NBQX (100 µM, 10 µl, Sigma), APV (100 µM, 10 µl, Sigma), L-glutamate (100 µM, 10 µl, Sigma), NMDA (100 µM, 10 µl, Sigma), WAY 100635 (100 µM, 10 µl, Sigma), and 8-OH-DPAT (100 µM, 10 µl, Sigma). These selected doses of drugs were used because previous studies have shown that glutamate and NMDA induce, while NBQX and APV attenuate, spinal long-term potentiation in anesthetized rats (45). Therefore, we adopted these effective doses without obtaining a dose-response curve in our study. Artificial cerebrospinal fluid [in mM: 118 NaCl, 3 KCl, 25 NaHCO3, 1.2 NaH2PO4, 1 MgCl2, 1.5 CaCl2, and 10 glucose (pH 7.4)] of identical volume to the tested agents was dispensed intrathecally to serve as a vehicle.

Data Analysis

All data in the text and figures are means ± SE. Statistical analysis of the reflex excitability between groups over the stimulation period was performed by means of ANOVA followed by a post hoc test. In all cases, a difference of P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Complete data were obtained from 35 rats. Under some conditions, animals were excluded from analysis, including three rats where the cannula tip for intrathecal application of drugs deviated by >0.5 mm from the target structure and two rats where the stimulation site at the pons varolli deviated >1 mm from the target structure. Therefore, there were 30 animals for statistical analysis. TS, RS, and glutamatergic agonist/antagonists were tested in all the animals. There were 10 rats for each test, including synchronized PS, serotonergic agonist injection, and serotonergic antagonist injection.

Electrical PS

As shown in Fig. 1B, after saline of subthreshold volume had been instilled into the urinary bladder via the transurethral bladder cannula, short-train pulses of PS produced a bladder contraction wave in the isovolumically filled urinary bladder characterized by an increase in IVP, indicating that the location of the stimulation electrodes was optimum. Then, the transurethral cannula was withdrawn. A midline abdominal incision exposed the urinary bladder and urethra, and an intravesical bladder cannula was inserted into the urinary bladder from the bladder dome so that the urinary bladder could drain freely. Train stimulation at the same site with identical parameters, when the bladder had drained, evoked no action potential in the EUS electromyogram (Fig. 1C).

Baseline Reflex Activity

Reflex activity evoked by afferent nerve TS (1/30 Hz) obtained from 1 of the 30 rats is shown in Fig. 1D. A single action potential was elicited by the TS. The mean latency for the TS to evoke the activity was 58.23 ± 8.41 ms (n = 30). The reflex activity varied little over a 30-min testing period (Fig. 2A). In addition, synchronized dorsolateral pontine tegmentum stimulation elicited no effects on TS-induced baseline reflex activity (data not shown).


Figure 2
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  		Fig. 2. Repetitive stimulation (RS)-induced reflex potentiation. A: single action potentials in the EUSE were evoked by TS. Afferent nerve RS evoked a longer-lasting reflex potentiation that was attenuated by 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline (NBQX; RS+NBQX) and abolished by D-2-amino-5-phosphonovalerate (APV; RS+APV). Glutamate (TS+Glu) or N-methyl-D-aspartic acid (NMDA; TS+NMDA) induces reflex potentiation of TS-induced reflex activity. The tracings before and after the break symbol show reflex activity at the onset of and at 30 min after stimulation onset, respectively. B: mean spike numbers (±SE, n = 30) in reflex activity induced by TS, RS, by TS combined with glutamate (TS+GLU) or NMDA (TS+NMDA) as well as by RS combined with NBQX (RS+NBQX) and APV (RS+APV). ** and ##: P < 0.01, significantly different from TS and RS, respectively.

 
RS-Induced Reflex Potentiation

A longer-lasting reflex potentiation was induced by afferent nerve RS (Fig. 2A, 1 Hz) at the same intensity as the TS. The evoked firing increased gradually following the onset of the RS, then reached a plateau at ~10 min and maintained this level until the cessation of stimulation. As shown in Fig. 2B, mean spike numbers evoked by the RS, counted at 30 min following the RS onset, increased significantly (16.12 ± 1.59 spikes/stimulation P < 0.01, n = 30) compared with the baseline reflex activity induced by the TS (1.00 ± 0.00 spike/stimulation).

Antagonism Caused by NBQX and APV

Intrathecal administration of NBQX (Fig. 2A, RS+NBQX, 100 µM, 10 µl) attenuated the RS -induced reflex potentiation. On the other hand, APV (RS+APV, 100 µM, 10 µl it) blocked the RS-induced reflex potentiation in the same preparations. Figure 2B shows that the mean spike numbers evoked by the afferent nerve stimulation, counted 30 min following stimulation onset, decreased significantly in both the RS with pretreated NBQX (RS+NBQX, 7.42 ± 0.57 spikes/stimulation, P < 0.01, n = 30) and in the RS with pretreated APV (RS+APV, 1.57 ± 0.29 spikes/stimulation, P < 0.01, n = 30) compared with the RS alone (16.12 ± 1.59 spikes/stimulation).

Agonist-Induced Potentiation

As shown in the top trace in Fig. 2A, single pulses of the TS on the afferent nerve evoked single action potentials. Intrathecal administration of glutamate (TS+GLU, 100 µM, 10 µl) and NMDA (TS+NMDA, 100 µM, 10 µl) both induced a longer-lasting reflex potentiation in TS-induced reflex activity (Fig. 2A, TS+GLU and TS+NMDA, respectively), which is similar to the RS-induced reflex potentiation. Mean spike numbers induced by the TS with intrathecal glutamate (Fig. 2B TS+GLU, 19.28 ± 1.10 spikes/stimulation, P < 0.01, n = 30) and by the TS with intrathecal NMDA (TS+NMDA, 16.86 ± 1.77 spikes/stimulation, P < 0.01, n = 30) both increased significantly, compared with the afferent nerve TS alone.

Facilitation Caused by PS

As shown in Fig. 3A, repetitive-train PS (1 Hz) evoked no action potential in reflex activity, while afferent nerve RS (1 Hz) induced a longer-lasting reflex potentiation. In addition, PS+RS enhanced the reflex potentiation that had been elicited by afferent nerve RS alone. Figure 3B summarizes the mean spike numbers induced by the afferent nerve TS (1.00 ± 0.00 spike/stimulation), the afferent nerve RS (16.12 ± 1.59 spikes/stimulation), by the train PS (0.00 ± 0.00 spike/stimulation) as well as RS+PS (25.17 ± 2.21 spikes/stimulation). The number of spikes elicited by RS+PS was significantly higher than that induced by the afferent fiber RS alone (25.17 ± 2.21 vs. 16.12 ± 1.59 spikes/stimulation, P < 0.01, n = 10).


Figure 3
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  		Fig. 3. Synchronized train PS facilitated RS-induced reflex potentiation. A: repetitive-train PS evoked no action potential in the EUSE. A long-lasting reflex potentiation was induced by afferent nerve RS. Synchronized train PS facilitated RS-induced reflex potentiation (PS+RS). The tracings before and after the break symbol show the reflex activities at the onset of and at 30 min after the stimulation onset. B: mean spike numbers (±SE, n = 30) induced by TS (bullet), RS ({blacktriangleup}), PS ({blacksquare}), and synchronized PS with the RS (RS+PS, {blacktriangledown}). ** and ##: P < 0.01, significantly different from activity induced by TS and RS, respectively.

 
Effects of Serotonin Agonist/Antagonist

As shown in Fig. 4A, a longer-lasting reflex potentiation was induced by afferent nerve RS. RS+PS produced facilitation in the RS-induced reflex potentiation. Intrathecal pretreatment with a serotonin antagonist, WAY 100635 (RS+PS+WAY, 100 µM, 10 µl), abolished the facilitation elicited by the synchronized train PS. Furthermore, an intrathecal catheter was inserted at the cervical (C3–C4) or thoracic (T5–T6) levels in each of three rats. In these cases, WAY 100635 showed no effect on the facilitation in reflex activities elicited by PS (data not shown). On the contrary, pretreatment with a serotonin agonist, 8-OH-DPAT (RS+DPAT, 100 µM, 10 µl), produced a similar facilitation in the RS-induced reflex potentiation as was evoked by the synchronized train PS. Figure 4B summarizes the mean spike numbers, at 30 min following the stimulation onset, induced by the afferent nerve TS (1.00 ± 0.00 spike/stimulation), by the afferent nerve RS (16.12 ± 1.59 spikes/stimulation), by PS (0.00 ± 0.00 spike/stimulation), by RS+PS (25.17 ± 2.21 spikes/stimulation), as well as the effects of WAY 100635 on the facilitation in reflex potentiation induced by the synchronized PS (RS+PS+WAY, 14.66 ± 1.58 spikes/stimulation, P < 0.01, n = 10) and 8-OH-DPAT (RS+DPAT, 26.16 ± 1.05 spikes/stimulation) on afferent RS. WAY 100635 significantly (P < 0.01, n = 10) reduced the number of spikes elicited by the synchronized train PS with the afferent fiber RS alone.


Figure 4
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  		Fig. 4. Effects of serotonergic agonist/antagonist on RS-induced reflex potentiation. A: pelvic afferent nerve RS induced a long-lasting reflex potentiation in EUSE activity. Synchronized train PS facilitated the RS-induced reflex potentiation (RS+PS). Intrathecal WAY 100635 abolished the facilitation caused by the synchronized train pontine stimulation (RS+PS+WAY). Intrathecal 8-OH-DPAT produced facilitation in RS-induced reflex potentiation without synchronized PS (RS+DPAT). The tracings before and after the break symbol show the reflex activities at the onset of and at 30 min after stimulation onset. B: mean spike numbers (±SE, n = 10) caused by TS, RS, PS, RS+PS, RS+PS+Way, and RS+DPAT. **, ##, and ++: P < 0.01, significantly different from activities induced by TS, RS, and RS+PS, respectively. No statistical significance (NS) is shown between RS and RS+PS+Way (P > 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our findings revealed that single-pulse pelvic afferent nerve stimulation evoked an action potential, whereas RS elicited long-lasting SRP. Synchronized PS superimposed on the afferent nerve RS produced facilitation in SRP. Serotonergic antagonists abolished the facilitatory effects elicited by the synchronized PS. Serotonergic agonists can elicit facilitation in the SRP without synchronized PS.

A repetitive activation of synaptic connections leads to activity-dependent reflex plasticity in brain areas (24, 38, 49). Nevertheless, forms of modulation on activity-dependent reflex plasticity are still being examined because they may help clarify the physiological relevance and the possibility of pharmacological manipulation in such a reflex plasticity (2, 48). In this study, we electrically stimulated the dorsolateral pontine tegmentum to modulate the SRP in the pelvic nerve-to-EUS reflex activity. We found that substantial changes in the SRP may be induced under physiological conditions. In addition, we also ascertained that intrathecal administration of WAY 100635, a selective silent 5-HT1A receptor antagonist, at the lumbosacral spinal cord abolished the facilitation on SRP that is a result of the dorsolateral pontine tegmentum stimulation. On the other hand, a selective 5-HT1A agonist, 8-OH-DPAT, produced facilitation in the SRP without synchronized PS. These results are consistent with previous reports that there is an excitatory role of spinal 5-HT1A receptors in the descending control of the lumbosacral spinal reflex as has been reported by Chang et al. (7, 8). Furthermore, not only is spinal reflex activity affected, our findings further demonstrate that the descending 5 HT1A system may modulate the SRP, i.e., a novel form of activity-dependent reflex plasticity at the lumbosacral level. These results imply that the 5-HT1A receptor agonist may provide a therapeutic target for spinal lumbosacral-mediated reflex urethra closure, which plays an important role in urine continence.

5-HT receptors are widely distributed in the central nervous system, including several areas involved in the functions of the urinary bladder and the urethra. The 5-HT1A and 5-HT1B receptors have been identified at the lumbosacral spinal cord, where preganglionic neurons innervating the urinary bladder and urethra are located (44, 56). The central 5-HT1A receptors exist as two functionally distinct populations. Somatodendritic autoreceptors are located on the somatic and dendrites of serotonergic neurons, whereas postsynaptic receptors are located on other neurons receiving serotonergic input. Owing to the differences between autoreceptor and postsynaptic 5-HT1A receptor populations, in terms of receptor reserve and receptor-effector coupling, several 5-HT1A receptor partial agonists have been identified, which act as antagonists at postsynaptic receptors and agonists at presynaptic receptors (19). However, this combination of effects of 5-HT1A receptor partial agonists can cause complex and conflicting pharmacological results. Cook et al. (13) and Fornal et al. (18) found WAY 100635 to be a potent, selective, and silent 5-HT1A receptor antagonist. We used WAY 100635 to evaluate the function of 5-HT1A receptors in the central nervous system. We found that intrathecal administration of WAY 100635 abolished the descending facilitation on SRP as a result of dorsolateral pontine tegmentum stimulation. On the contrary, WAY 100635 had no effect on the descending facilitation in SRP at the cervical or thoracic levels of the spinal cord. These findings indicate that for WAY 100635 to affect SRP in the pelvic nerve-to-EUS reflex activity, it must be administered intrathecally at the L6-S1 spinal cord level. In addition, intrathecal administration of the 5-HT1A-receptor agonist 8-OH-DPAT facilitated SRP without synchronized PS and thus could mimic the facilitated descending serotonergic modulation on the SRP. Although giving an agonist might produce a nonphysiological state, these results, at least in part, indicate that spinal 5-HT1A receptors at the L6-S1 level play an important role in the descending modulation on SRP, which is important in urine continence.

Reflex urethra closure mechanisms have not been clarified thoroughly, especially in small animals used in experiments, including rats. The clarification of these mechanisms may be very important for the development of pharmacotherapy for urinary disorders (14, 15). During the initial stage when a voiding contraction occurs, in some animal such as rats, the intravesical pressure gradually increases without urine emission. During this period, the mechanoreceptors on the stretched bladder wall are excited and generate impulses, which are transmitted to the spinal cord through the pelvic afferent nerve. After integration within the spinal cord, motor impulses are conducted centrifugally to the EUS via the pudendal efferent nerves. These impulses, therefore, induce EUS contraction to establish sufficient intravesical pressure for urine propulsion (11, 29). Recent studies investigating SRP in pelvic nerve-to-EUS reflex activity have demonstrated that not only the pelvic nerve-to-EUS reflex activity itself but also the stimulation-induced SRP in such a reflex may play an important role in the urethra closure mechanism (31–36). In the present study, superimposed pontine tegmentum stimulation produced facilitation in RS-induced SRP in pelvic nerve-to-EUS reflex activity, which suggests that descending modulation coming from the pontine tegmentum may also be essential for the physiological urethra closure functions.

Investigations on the neural control of the lower urinary tract including the urinary bladder (53, 59, 60, 62) and urethra (7, 61) have revealed the involvement of glutamatergic NMDA-dependent neurotransmission in the lumbosacral cord mediating micturition functions. It is widely accepted that glutamatergic NMDA neurotransmission underlies activity-dependent reflex plasticity (52). In this study, we induced a glutamate-dependent SRP by repetitive electric shocks. We found that NMDA and AMPA receptor antagonists attenuated the SRP in the pelvic nerve-to-EUS reflex activities. Our findings suggest that RS-induced SRP may share a neurotransmission similar to the well-investigated long-term potentiation (12, 21, 26, 39). This preliminary finding is in accordance with a recent report by Randic et al. (45). They reported that the strength of glutamatergic primary afferent transmission in the spinal dorsal horn neurons might be potentiated following tetanic peripheral inputs. However, in the present study, we used the multiple fiber recording technique; therefore, whether this enhancement is mediated by a "long-term potentiation-like" synaptic transmission needs further investigation of the synaptic efficacy on the dorsal horn neurons within the spinal cord.

As shown in the Fig. 1B, train pulse PS induced a contraction in an isovolumic filled urinary bladder, which drove firing in the EUS electromyogram. This might suggest that the sacral parasympathetic nucleus may interact with, or have a neural connection to, Onuf's nucleus that innervates the external urethra sphincter through the somatic pudendal nerve (Fig. 5). To verify this mechanism, further investigation using the single-neuron recording technique is necessary.


Figure 5
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  		Fig. 5. Diagram showing putative pathway for pelvic nerve-to-urethra reflex potentiation as well as the descending modulation coming from the dorsolateral pontine tegmentum. A: neural connection among pelvic afferent coming from urinary bladder, pudendal efferent nerve arising from Onuf's nucleus, and the descending innervation from the pons varolli. B: possible neurotransmitters involved in this putative circuitry. The dashed pathway indicates that whether the reflex potentiation is mediated by a "long-term potentiation-like" transmission or polysynaptic pathway is still unclear. In addition, whether the sacral parasympathetic nucleus may either have an interaction with, or a neural connection to, Onuf's nucleus is also unknown.

 

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This research was supported by grants from the National Science Council in Taiwan (NSC-93-2314-B-040-013 and NSC-94-2314-B-040-026) to G. D. Chen and (94-2320-B-040-026 and 95-2320-B-040-026) to T. B. Lin.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T.-B. Lin, Dept. of Physiology, College of Medicine, Chung-Shan Medical Univ., No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung, 40201, Taiwan (e-mail: tblin{at}csmu.edu.tw)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* K.-C. Tung and T.-B. Lin contributed equally to this study. Back


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