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ORIGINAL RESEARCH—BASIC SCIENCE: Effect of the Destruction of Cells Containing the Serotonin Reuptake Transporter on Urethrogenital Reflexes
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ORIGINAL RESEARCH—BASIC SCIENCE Effect of the Destruction of Cells Containing the Serotonin Reuptake Transporter on Urethrogenital Reflexes Karla Gravitt, BSc, and Lesley Marson, PhD Division of Urology, Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA DOI: 10.1111/j.1743-6109.2007.00436.x
ABSTRACT
Introduction. The urethrogenital (UG) reflex is an autonomic and somatic response that mimics some of the physiological changes seen during ejaculation. The UG reflex is tonically inhibited by neurons in the ventral medulla, an area containing serotonin neurons. Aim. To examine the effect of lesions of brain neurons containing the serotonin reuptake transporter (SERT) on ejaculatory-like reflexes. Methods. Anti-SERT saporin (80 nL, 1 mM) or saline was injected bilaterally into the ventrolateral medulla of male Sprague–Dawley rats. Ten to 18 days later, animals were deeply anesthetized and the presence of the UG reflex was examined before and after acute spinal cord transection (T9–10). Following the experiment the presence and number of serotonin and norepinephrine containing neurons (using tryptophan hydroxylase and dopamine beta-hydroxylase, respectively) was performed. Main Outcome Measures. The UG reflex and cell counts. Results. In saline-injected controls the UG reflex was not evoked in the anesthetized, intact preparation, indicating the presence of the supraspinal inhibition, as previously reported. Injection of anti-SERT saporin into the ventrolateral medulla allowed the UG reflex to be activated in the intact preparation, thus removed the inhibition. This was associated with a decrease in the number of serotonin neurons in the ventrolateral medulla and raphe. No change in the number of noradrenergic neurons was observed. Conclusion. These studies suggest that ventral medullary neurons containing SERT are involved in the tonic inhibition of the UG reflex. Gravitt K, and Marson L. Effect of the destruction of cells containing the serotonin reuptake transporter on urethrogenital Reflexes. J Sex Med 2007;4:322–331. Key Words. Ejaculation; Serotonin; Medulla
Introduction
E
jaculatory and erectile reflexes are regulated by spinal circuits that receive modulatory inputs from the brain [1]. The urethrogenital (UG) reflex model is an autonomic and somatic response that is seen upon stimulation of the urethra via the pudendal sensory nerve, in the anesthetized, spinalized rat and comprises of rhythmic firing of the pudendal motor, pelvic and hypogastric nerves, and expulsion of the urethral
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contents [1,2]. As similar changes are observed during ejaculatory responses in humans, this model has been proposed to mimic some of the physiological changes that accompany ejaculation [1,3,4]. This model has also proven fundamental in furthering our understanding of pelvic responses in that elucidation of both inhibitory and facilitatory brain regions that regulate the UG reflex have been identified [5–7]. One area reported to mediate an inhibition of ejaculatory-like responses is the nucleus paragigantocellularis (nPGi), which © 2007 International Society for Sexual Medicine
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Anti-SERT Saporin Lesion of VLM and UG Reflex is located in hindbrain in the ventrolateral medulla (VLM). Lesions of the VLM release the spinal inhibition of the UG reflex, facilitate ex copula sexual reflexes, and increase sexual function in exhaustion models [7,8]. Serotonin-containing neurons and serotonin receptors are located in the VLM, in regions that overlap with sites thought to mediate this inhibition, as well as in the midline raphe [1,8,9]. In addition, serotonin neurons originating in the VLM innervate spinal circuits that regulate pelvic organ function [8,10–15]. Selective serotonin reuptake inhibitors (SSRIs) given to treat psychological disorders such as depression and anxiety often cause reduced sexual function, including decreased arousal and anorgasmia [16,17]. SSRIs act on serotonin reuptake mechanisms via the serotonin reuptake transporter (SERT) that mediates inactivation of serotonin following its release at synapses. Inhibition of the serotonin reuptake system results in increased availability of serotonin that is thought to mediate the delayed ejaculation in humans [16,17]. Studies supporting this hypothesis have demonstrated that peripheral administration of serotonin may act via 5-hydroxytryptamine (5-HT)1B and 5-HT2C receptors to inhibit ejaculatory behavior [8,16,18–21]. However, facilitation of ejaculation through 5-HT1A receptors has also been reported [18,22]. Previous studies demonstrated that intrathecal or intracerebroventicular administration of 5,7 dihydroxytryptamine (5,7 DHT) removed the descending inhibition of the UG reflex, which was associated with a significant decrease in serotonin levels in the spinal cord [23]. The origin of spinal serotonergic inputs arises primarily from neurons in the pons and medulla. In order to further understand the link between SSRIs and ejaculatory-like responses, the present study examined the effect of lesions of ventral medullary neurons containing the SERT on the UG reflex. Removal of neurons containing the SERT receptor was achieved using the ribosomeinactivating protein, saporin, which was conjugated to a monoclonal antibody to the fourth extracellular domain of the SERT (Advanced Targeting Inc.) [24]. Saporin conjugates act as targeted neurotoxins that result in selective death of neurons containing the appropriate receptors [24–27]. If sexual reflexes are influenced by saporin removal of brain neurons containing the SERT, this would further confirm a role for central serotonin in regulating ejaculatory-like reflexes. This technique has proven successful in studying the
central nervous system control of respiration and bladder function [28–30]. Methods
Anti-SERT Saporin Injections All experimental procedures involving animals were approved by the University of North Carolina Institutional Animal Care and Use Committee in accordance with National Institute of Health guidelines. Male Sprague–Dawley rats (N = 22) were anesthetized with a mixture of ketamine and xylazine (50 mg/kg + 4 mg/kg, intraperitoneally, respectively) and placed into a stereotaxic apparatus. A craniotomy was performed to allow access to the nPGi using aseptic techniques. A Hamilton syringe (1 mL) was lowered into the rostral nPGi (coordinates 2.4 mm caudal to lambda, 1.2 mm lateral to the midline, and 8.5–8.75 mm deep) and anti-SERT saporin (anti-SERT SAP; Advanced Targeting Systems, San Diego, CA, USA, 80 nL, 1 mM) was slowly injected into the brain. Four control animals received vehicle injections of saline (80–100 nL, containing 0.1% pontamine sky blue). The syringe remained in place for 5 minutes after the end of the injection then was slowly removed. Injections were made bilaterally. The craniotomy was sealed with bone wax and the skin overlying the skull was sutured. Three additional control animals received no injections. Physiology Following a recovery period of 10–18 days [24,31], all rats were anesthetized with urethane (1.2– 1.4 g/kg intraperitoneally). The carotid artery and jugular vein were cannulated for measurement of arterial pressure and administration of drugs, respectively. Rats were maintained at ~38°C with a thermostatically controlled heating blanket. A catheter (PE 50), connected to an infusion pump and a pressure transducer, was inserted into the urethra via a bladder incision and tied in place. Bipolar silver electrodes were used to obtain electromyographic (EMG) recordings from the bulbospongiosus muscles. Recording signals were fed into a high impedance preamplifier, displayed onto a polygraph and oscilloscope, and fed into an audio monitor and Computer (PowerLab 8 and Chart 5.0 from AD Instruments, Colorado Springs, CO, USA). Raw EMG signals were rectified and integrated with a 100-millisecond time constant. Infusion of saline (0.4 mL/min) into the urethra and brief occlusion of the urethral meatus was used to J Sex Med 2007;4:322–331
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evoke the UG reflex (poststimulus rhythmic contractions of the bulbospongiosus muscles) [2]. The presence or absence of the UG reflex was examined in the intact preparation. Subsequently, the spinal cord was cut at T9–10 and after at least 30 minutes the presence of the UG reflex was re-examined.
Immunohistochemistry Immediately following physiological recordings rats were perfused with 4% paraformaldehyde while under deep anesthesia. Brain tissue was removed and placed in 4% paraformaldehyde overnight and then transferred to 30% sucrose. Coronal sections (35 mm) of the brainstem were cut and transferred to cyroprotectant [32]. A series of sections (every 0.2 mm) was processed for immunohistochemical staining of tryptophan hydroxylase (TPOH, the rate-limiting enzyme involved in the synthesis of serotonin) in order to examine the number and location of serotonin-containing neurons. Separate sections (every 0.4 mm) were incubated with dopamine beta-hydroxylase (DBH) in order to examine the number and location of norepinephrine- and epinephrine-containing neurons. Free floating sections were rinsed in potassium phosphate-buffered saline and then incubated overnight with either a mouse antibody against TPOH (1:5,000; Sigma, St. Louis, MI, USA), or a mouse antibody against DBH (1:1,000; Chemicon, Temecula, CA, USA). After washing, sections were incubated in biotinylated rabbit anti-mouse immunoglobulin G (1:500; Vector Laboratories, Burlingame, CA, USA). Sections were then incubated in avidin–biotin complex (ABC Elite 1:1,000; Vector Laboratories) and reacted with diaminobenzidine solution (Vector Laboratories). All sections were mounted on plusR slides, and coverslipped. A series of sections were mounted and counterstained with thionin in order to visualize the injection sites. Data Analysis The numbers of immunostained cells in the raphe pallidus, raphe obscurus, raphe magnus, ventral surface, parapyramidal region, and the VLM (Figure 1) were counted in each section. The caudal extent of the inferior olives was designated as 0 and the section where the trapezoid bodies crossed the midline was found at ~3 mm rostral. In addition, the number of immunostained neurons in the A5, and caudal median raphe and dorsal raphe area were counted [33]. The person countJ Sex Med 2007;4:322–331
Figure 1 Diagrammatic representations of the medulla indicating the locations in which tryptophan hydroxylase (TPOH)-positive cells were counted. Cells stained overlying the pyramids (pp) were included in the raphe magnus (RM) counts more rostrally. These sections represent approximately 0.6 mm and 2.4 mm from the caudal extent of the inferior olives. Amb = nucleus ambiguus; FN = facial nucleus; IO = inferior olives; NTS = nucleus tractus solitarius; p = pyramids; pp = parapyramidal region; Rp = raphe pallidus; RM = raphe magnus; RO = raphe obscurus; Ve = vestibular nucleus; VLM = ventrolateral medulla; VS = ventral surface.
ing the cells was blinded to the treatment group. The number of immunostained cells in each area was compared between groups of animals treated with anti-SERT SAP that demonstrated the UG reflex in the spinally intact preparation and controls that did not demonstrate the UG reflex in the intact preparation. Statistical significance (P < 0.05) was determined with a one-way anova followed by Scheffe’s post hoc tests using SPSS 13.0 for Windows. Results
Physiology Three animals (one saline and two anti-SERT SAP) were excluded from the data analysis because of physical damage from the injections that was found upon subsequent histological examination. In spinally intact controls (saline-injected and noninjected) the UG reflex could not be evoked (Figure 2, control, intact). However, after cutting the spinal cord, rhythmic contractions of the bulbospongiosus muscles were reliably evoked at low thresholds (25–40 mm Hg) in response to urethral distension (Figure 2, control, spinalized).
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Figure 2 Effect of VLM injections of anti-SERT SAP on the UG reflex. Saline (control) or anti-SERT SAP was injected bilaterally into the VLM 14 days before recording the UG reflex. In controls with intact spinal cord the UG reflex could not be evoked. Pretreatment with anti-SERT SAP exposed the UG reflex in the intact preparation (anti-SERT SAP, intact). After spinal cord transection at T10 (spinalized) the UG reflex was present in both the control and antiSERT SAP animals. The UG reflex can be seen as rhythmic firing of the bulbospongiosus muscles (BS EMG) following release of the occlusion (black lines at bottom of urethral pressure trace show the occlusion period). BS EMG was rectified and integrated with a 200-millisecond time constant.
In contrast, the UG reflex was present in eight animals (62%) that received anti-SERT SAP injections before the spinal cord was transected (Figure 2, anti-SERT SAP, intact). In these animals the UG reflex was consistently evoked at low thresholds (25–55 mm Hg) and the latency to initiation of the UG reflex was 41 ⫾ 6.9 seconds (range 10–65 seconds) (Figure 2, anti-SERT SAP, intact). The latency to the UG reflex after spinalization was significantly shorter than before spinal cuts in both the control and anti-SERT SAP groups (Figure 2, anti-SERT SAP, spinalized). The remaining animals (38%) that received antiSERT SAP showed either inconsistent responses or no rhythmic contractions of the bulbospongiosus muscles prior to spinal cord transection.
Immunohistochemistry TPOH Staining In the medulla the majority of TPOH-positive cells were found in the raphe obscurus and raphe magnus at 0–2 mm, and 2–3 mm, respectively (Figure 3). TPOH-containing cells in the raphe pallidus were located primarily 0.2–2.2 mm at the level of the inferior olives (Figure 3). TPOHpositive cells in the VLM included cells lateral to the pyramidal tract, on the ventral surface of the medulla, and in the parapyramidal region (cells lying over the pyramids in the gigantocellular reticular formation), as shown in Figure 1. In the VLM the majority of cells staining for TPOH were found between 1.6 and 2.8 mm; this area
overlaps with the rostral nPGi [33]. At 2–3 mm the parapyramidal cells formed an extension of the raphe magnus and thus TPOH-containing cells at these levels were included in the raphe magnus counts. This area consists of the B3 region of the serotonergic neurons [34]. TPOH-positive cells were also found in the median raphe and dorsal raphe area. Cell counts in animals that received anti-SERT SAP and displayed UG reflexes in the spinally intact preparation were compared with controls. Presence of the UG reflex in the intact preparation was associated with a significant decrease in TPOH-positive cells in the raphe magnus, raphe obscurus, raphe pallidus, and VLM (Figures 3 and 4). A reduction in the number of TPOH-positive cells was found throughout the length of the medulla in the raphe obscurus and raphe magnus. In the VLM a significant decrease in the number of TPOH-containing cells was also found, particularly from 2.0 to 3.2 mm at the level of and rostral to the injection site (Figures 3 and 4). However, there was no difference between groups in the numbers of TPOH cells counted on the ventral surface, in the parapyramidal region, the median raphe or dorsal raphe (Figure 5). Animals that received anti-SERT SAP and showed either inconsistent or no rhythmic contractions of the bulbospongiosus muscles prior to spinal cord transection had variable cells counts that were on average not different from controls. J Sex Med 2007;4:322–331
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Figure 3 Graphs illustrating the rostrocaudal distribution of tryptophan hydroxylase (TPOH)-positive cells in the raphe pallidus, raphe obscurus + raphe magnus, ventrolateral medulla, and total cell counts. Each data point is the average of the total number of cells counted at 0.2 mm intervals (the bars show the SE of the mean). Controls (solid lines, filled circles) and antiSERT saporin group (dashed lines, open circles) that displayed the UG reflex in the spinally intact preparation are plotted. N = 4–7.
DBH Staining Dopamine beta-hydroxylase-containing neurons were found in the VLM and in the A5 region. In the caudal medulla (0–1.8 mm) an average of 39 ⫾ 12 and 34 ⫾ 11 DBH-positive cells per section were found in the control and anti-SERT SAP groups, respectively. In the A5 region 32 ⫾ 10 and 27 ⫾ 11 DBH-positive cells per
Figure 4 Histogram illustrating the total number of tryptophan hydroxylase (TPOH)-positive cells counted in the medulla in controls (open bars) and the anti-SERT SAP group that displayed the UG reflex in the spinally intact preparation (solid bars). RO = raphe obscurus; RM = raphe magnus; RP = raphe pallidus; VLM = ventrolateral medulla; VS = ventral surface; PP = parapyramidal region; MR = median raphe; DR = dorsal raphe. N = 5–7. *P < 0.05, **P < 0.001 indicate a significant difference between the control and anti-SERT SAP group.
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section were found in the control and anti-SERT SAP groups, respectively. There were no significant differences between the groups.
Discussion
Partial reduction of serotonin containing neurons in the medullary raphe and VLM allowed the elicitation of the UG reflex in the anesthetized spinally intact preparation. The associated decrease in TPOH staining in the VLM with the appearance of perineal muscle activity is consistent with previous studies that suggested that descending serotonergic inputs from the nPGi to the lumbosacral spinal cord are involved in mediating the supraspinal inhibition of ejaculatory-like responses [35–37]. Lesions of the ventral medulla in the region of the nPGi removed the supraspinal inhibition of the UG reflex, facilitated ex copula penile reflexes, and improved sexual behavior in sexually exhausted rats. Stimulation of the nPGi also alters pudendal motor output and produces inhibition of penile reflexes, and neurons in this region receive convergent information from multiple areas including the penis and UG tracts [38–40]. Serotonergic neurons in the VLM project directly to pudendal motor neurons and preganglionic neurons that are activated in response to UG stimulation [7,14,15,41–46]. Taken together these data indicate that brainstem-spinal serotoner-
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Figure 5 Photomicrographs showing tryptophan hydroxylase (TPOH) neurons in the ventrolateral medulla (A and B, at 2 mm level) and raphe magnus (C and D, at 2.6 mm level) in control (A and C) and anti-SERT saporin injected (B and D) animals. Note: more TPOH-labeled cells in the controls. Scale bar = 60 mm.
gic pathways participate in the regulation of ejaculatory-like responses including the UG reflex. The number of TPOH-containing neurons in the anti-SERT SAP group was also reduced in the medullary raphe. Previous studies have suggested that the raphe obscurus, raphe pallidus, and dorsal raphe may be involved in sexual function [47]. Lesions of the raphe obscurus decreased the number of intromissions and ejaculations [47]. However, the occurrence of penile reflexes was not affected by raphe obscurus lesions [42]. Neurons containing the SERT are also present in the median raphe and dorsal raphe and the dorsal raphe is sensitive to gonadal steroid hormone treatment, suggesting this nucleus may also be involved in sexual function. In one study, large injections of 5,7 DHT into the dorsal raphe decreased ejaculatory latency in awake sexual behavior tests [41]. However, no changes in the numbers of TPOH neurons were found in the median raphe and dorsal raphe in the present study suggesting that these areas are not essential for the supraspinal inhibition of the UG reflex. Very few studies have examined brainstem changes in neural activity that is associated with sexual behavior. One study reported significant changes in the number of c-fos-activated neurons in the VLM and raphe pallidus after ejaculation
that was thought to be related to the postejaculatory refractory period [43]. Because of the close proximity of the medullary raphe and the VLM and their known reciprocal projections, lesions of or injections into one site may affect the function of the other [48–51]. In addition, SERT is found primarily on axons, dendrites, and terminals of serotonin neurons [52], therefore loss of serotonergic raphe neurons in the present study may have occurred through raphe projections to the VLM. No decrease in TPOH-positive cell numbers on the ventral surface and caudal parapyramidal regions was found in the present study suggesting that these serotonergic neurons are not involved in tonic inhibition of the UG reflex. Many but not all the spinally projecting neurons in the ventral medulla contain serotonin [8–10,53,54]. In addition, other neurotransmitters innervate the lumbosacral spinal cord. In the present study no change in the number of DBHcontaining neurons was found suggesting that the saporin injections did not affect norepinephrine and epinephrine neurons. Other neurotransmitters, for example, substance P and thyrotropinreleasing hormone containing neurons, also project to the spinal cord and these neurotransmitters are colocalized within a distinct proportion of serotonergic neurons [9,54–56]. Therefore, destruction of neurons containing the SERT may J Sex Med 2007;4:322–331
328 also affect other neurotransmitters colocalized in these neurons. Further studies are required to investigate the role of these multiple neurotransmitters in the control of sexual function. Examination of spontaneous, rather than reflex-induced, erectile and ejaculatory responses in rats has been conducted using pchloroamphetamine (PCA), which results in the release catecholamines and serotonin from neurons [57]. These studies demonstrated spontaneous ejaculatory-like responses after systemic administration of PCA in both awake and anesthetized models [58–61], suggesting that release of monoamines can facilitate or induce spontaneous ejections and ejaculation. While PCA can act in the brain to produce its effects on sexual behavior, evidence for a spinal site of action has also been provided [57,58,62–64]. Systemic administration of PCA also acts on peripheral organs and nerves and a recent study demonstrated that ejaculatory responses induced by PCA require a peripheral-spinal pathway [65]. While it is generally thought that the PCA effects on sexual reflexes are via the serotonin system, PCA results in an increase in catecholamines, including dopamine, which has a potent effect on facilitating sexual responses [66,67]. In addition, activation of 5-HT1A receptors facilitate ejaculation while activation of 5-HT1B/2C inhibit sexual responses, thus the resultant effect on ejaculatory reflexes may depend on the receptors predominately activated [18–20]. Therefore, multiple serotonin pathways (e.g., brain, brain-spinal, and peripheral-spinal), receptors, and mechanisms (inhibitory and facilitatory) are involved in regulating erectile and ejaculatory events. Previous studies have successfully used saporin conjugates, including anti-SERT SAP, to selectively destroy neurotransmitter-specific cells in the brain [24,28–30,68]. The present study concurs with these studies in that saporin conjugates can selectively destroy specific neurotransmitter populations. However, further studies are required to investigate the effect on neurons containing multiple neurotransmitters.
Acknowledgments
The authors would like to thank Dr. Rong-S Cai for his expert technical assistance. Corresponding Author: Lesley Marson, PhD, The University of North Carolina, Division of Urology, Department of Surgery, 4024 Burnett Womack BuildJ Sex Med 2007;4:322–331
Gravitt and Marson ing, Chapel Hill, NC 27599, USA. Tel: (919) 966-9252; Fax: (919) 966-0098; E-mail: [email protected] Conflict of Interest: None declared. References
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330 43 Hamson DK, Watson NV. Regional brainstem expression of Fos associated with sexual behavior in male rats. Brain Res 2004;1006:233–40. 44 Holmes GM. 5-Hydroxytryptamine2C receptors on pudendal motoneurons innervating the external anal sphincter. Brain Res 2005;1057:65–71. 45 Tanaka J, Arnold AP. An electrophysiological study of descending projections to the lumbar spinal cord in adult male rats. Exp Brain Res 1993;96:117– 24. 46 Zhuo M, Gebhart GF. Tonic cholinergic inhibition of spinal mechanical transmission. Pain 1991; 46:211–22. 47 Yamanouchi K, Kakeyama M. Effect of medullary raphe lesions on sexual behavior in male rats with or without treatments of p-chlorophenylalanine. Physiol Behav 1992;51:575–9. 48 Andrezik JA, Chan-Palay V, Palay SL. The nucleus paragigantocellularis lateralis in the rat. Demonstration of afferents by the retrograde transport of horseradish peroxidase. Anat Embryol (Berl) 1981;161:373–90. 49 Van Bockstaele EJ, Biswas A, Pickel VM. Topography of serotonin neurons in the dorsal raphe nucleus that send axon collaterals to the rat prefrontal cortex and nucleus accumbens. Brain Res 1993;624:188– 98. 50 Dampney RA, Czachurski J, Dembowsky K, Goodchild AK, Seller H. Afferent connections and spinal projections of the pressor region in the rostral ventrolateral medulla of the cat. J Auton Nerv Syst 1987;20:73–86. 51 Hermann DM, Luppi PH, Peyron C, Hinckel P, Jouvet M. Afferent projections to the rat nuclei raphe magnus, raphe pallidus and reticularis gigantocellularis pars alpha demonstrated by iontophoretic application of choleratoxin (subunit b). J Chem Neuroanat 1997;13:1–21. 52 Huang J, Pickel VM. Serotonin transporters (SERTs) within the rat nucleus of the solitary tract: Subcellular distribution and relation to 5HT2A receptors. J Neurocytol 2002;31:667–79. 53 Skagerberg G, Bjorklund A. Topographic principles in the spinal projections of serotonergic and nonserotonergic brainstem neurons in the rat. Neuroscience 1985;15:445–80. 54 Nevin K, Zhuo H, Helke CJ. Neurokinin A coexists with substance P and serotonin in ventral medullary spinally projecting neurons of the rat. Peptides 1994;15:1003–11. 55 Sasek CA, Wessendorf MW, Helke CJ. Evidence for co-existence of thyrotropin-releasing hormone, substance P and serotonin in ventral medullary neurons that project to the intermediolateral cell column in the rat. Neuroscience 1990;35:105–19. 56 Marson L. Evidence for colocalization of substance P and 5-hydroxytryptamine in spinally projecting neurons from the cat medulla oblongata. Neurosci Lett 1989;96:54–9. J Sex Med 2007;4:322–331
Gravitt and Marson 57 Fuller RW. Effects of p-chloroamphetamine on brain serotonin neurons. Neurochem Res 1992; 17:449–56. 58 Yonezawa A, Watanabe C, Ando R, Furuta S, Sakurada S, Yoshimura H, Iwanaga T, Kimura Y. Characterization of p-chloroamphetamine-induced penile erection and ejaculation in anesthetized rats. Life Sci 2000;67:3031–9. 59 Renyi L. Ejaculations induced by pchloroamphetamine in the rat. Neuropharmacology 1985;24:697–704. 60 Humphries CR, O’Brien M, Paxinos G. PCA: Effects on ejaculation, thermoregulation, salivation, and irritability in rats. Pharmacol Biochem Behav 1980;12:851–4. 61 Humphries CR, Paxinos G, O’Brien M. Mechanisms of PCA-induced hypothermia, ejaculation, salivation and irritability in rats. Pharmacol Biochem Behav 1981;15:197–200. 62 Stafford SA, Bowery NG, Tang K, Coote JH. Activation by p-chloroamphetamine of the spinal ejaculatory pattern generator in anaesthetized male rats. Neuroscience 2006;140:1031–40. 63 Stafford SA, Tang K, Coote JH. Sympathetic genital responses induced by p-chloroamphetamine in anaesthetized female rats. Neuroscience 2006; 138:725–32. 64 Moorman JM, Leslie RA. P-chloroamphetamine induces c-fos in rat brain: A study of serotonin2A/2C receptor function. Neuroscience 1996;72:129–39. 65 Yonezawa A, Yoshizumi M, Ebiko M, Iwanaga T, Kimura Y, Sakurada S. Evidence for an involvement of peripheral serotonin in p-chloroamphetamineinduced ejaculation of rats. Pharmacol Biochem Behav 2005;82:744–50. 66 Hull EM, Muschamp JW, Sato S. Dopamine and serotonin: Influences on male sexual behavior. Physiol Behav 2004;83:291–307. 67 Argiolas A, Melis MR. The neurophysiology of the sexual cycle. J Endocrinol Invest 2003;26:20–2. 68 Fargo KN, Sengelaub DR. Exogenous testosterone prevents motoneuron atrophy induced by contralateral motoneuron depletion. J Neurobiol 2004; 60:348–59. Commentary on Gravitt K, Marson L. Effect of the Destruction of Cells Containing the Serotonin Reuptake Transporter on Urethrogenital Reflexes
In the last decade, efforts have been made to investigate the neurobiology of ejaculation and in parallel pharmacological management of premature ejaculation (PE) has been proposed. Selective serotonin reuptake inhibitors (SSRIs) used to treat depression have long been known to cause sexual dysfunctions, notably delayed ejaculation and sometimes anejaculation. Based on this
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Anti-SERT Saporin Lesion of VLM and UG Reflex observation, numerous clinical studies have investigated the potential interest of chronic antidepressant SSRIs in lengthening ejaculation time in PE patients [1] and, as recently reported, on demand treatment with the short-acting SSRI dapoxetine appears effective and safe [2]. However, the mechanism of action by which SSRIs delay ejaculation is still not fully understood [3]. The present study is a contributive effort made to clarify this issue. Using the urethrogenital reflex model in anesthetized rats coupled with a highly specific neutotoxic lesioning approach, the authors showed the primary role of serotonergic neurons expressing serotonin transporter and located in the ventrolateral medulla and medullary raphe on the control of the ejaculatory motor reflex. Although the experimental paradigm used here is extra physiological and mimics only some aspects of the ejaculatory response, it has been previously shown useful in improving our understanding on the control of perineal striated muscles activity, a key element in the expulsion phase of ejaculation. Nonselective electrolytic lesion of the paragigantocellular nucleus, which includes ventrolateral medulla, has been shown to reverse fluoxetine-delaying effect on ejaculation in male rats [4], tending to give a physiological meaning for the present findings. However, this remains to be confirmed in behavioral investigations using the elegant and selective approach described here. Finally, it is to
be noticed that the present results provide indications for the differentiation of the mechanism of action between antidepressant and ejaculationretarding properties of SSRIs. François Giuliano, MD, PhD Department of Neuro-Uro-Andrology Physical Medicine and Rehabilitation, Raymond Poincare Hospital, Garches France E-mail: [email protected]
References
1 Althof SE. Prevalence, characteristics and implications of premature ejaculation/rapid ejaculation. J Urol 2006;175(3 Pt 1):842–8. 2 Pryor JL, Althof SE, Steidle C, Rosen RC, Hellstrom WJ, Shabsigh R, Miloslavsky M, Kell S, Dapoxetine Study Group. Efficacy and tolerability of dapoxetine in treatment of premature ejaculation: An integrated analysis of two double-blind, randomised controlled trials. Lancet 2006;368:929–37. 3 Giuliano F, Clement P. Serotonin and premature ejaculation: From physiology to patient management. Eur Urol 2006;50:454–66. 4 Yells DP, Prendergast MA, Hendricks SE, Nakamura M. Fluoxetine-induced inhibition of male rat copulatory behavior: Modification by lesions of the nucleus paragigantocellularis. Pharmacol Biochem Behav 1994;49:121–7.
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ORIGINAL RESEARCH—BASIC SCIENCE Effect of the Destruction of Cells Containing the Serotonin Reuptake Transporter on Urethrogenital Reflexes Karla Gravitt, BSc, and Lesley Marson, PhD Division of Urology, Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA DOI: 10.1111/j.1743-6109.2007.00436.x
ABSTRACT
Introduction. The urethrogenital (UG) reflex is an autonomic and somatic response that mimics some of the physiological changes seen during ejaculation. The UG reflex is tonically inhibited by neurons in the ventral medulla, an area containing serotonin neurons. Aim. To examine the effect of lesions of brain neurons containing the serotonin reuptake transporter (SERT) on ejaculatory-like reflexes. Methods. Anti-SERT saporin (80 nL, 1 mM) or saline was injected bilaterally into the ventrolateral medulla of male Sprague–Dawley rats. Ten to 18 days later, animals were deeply anesthetized and the presence of the UG reflex was examined before and after acute spinal cord transection (T9–10). Following the experiment the presence and number of serotonin and norepinephrine containing neurons (using tryptophan hydroxylase and dopamine beta-hydroxylase, respectively) was performed. Main Outcome Measures. The UG reflex and cell counts. Results. In saline-injected controls the UG reflex was not evoked in the anesthetized, intact preparation, indicating the presence of the supraspinal inhibition, as previously reported. Injection of anti-SERT saporin into the ventrolateral medulla allowed the UG reflex to be activated in the intact preparation, thus removed the inhibition. This was associated with a decrease in the number of serotonin neurons in the ventrolateral medulla and raphe. No change in the number of noradrenergic neurons was observed. Conclusion. These studies suggest that ventral medullary neurons containing SERT are involved in the tonic inhibition of the UG reflex. Gravitt K, and Marson L. Effect of the destruction of cells containing the serotonin reuptake transporter on urethrogenital Reflexes. J Sex Med 2007;4:322–331. Key Words. Ejaculation; Serotonin; Medulla
Introduction
E
jaculatory and erectile reflexes are regulated by spinal circuits that receive modulatory inputs from the brain [1]. The urethrogenital (UG) reflex model is an autonomic and somatic response that is seen upon stimulation of the urethra via the pudendal sensory nerve, in the anesthetized, spinalized rat and comprises of rhythmic firing of the pudendal motor, pelvic and hypogastric nerves, and expulsion of the urethral
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contents [1,2]. As similar changes are observed during ejaculatory responses in humans, this model has been proposed to mimic some of the physiological changes that accompany ejaculation [1,3,4]. This model has also proven fundamental in furthering our understanding of pelvic responses in that elucidation of both inhibitory and facilitatory brain regions that regulate the UG reflex have been identified [5–7]. One area reported to mediate an inhibition of ejaculatory-like responses is the nucleus paragigantocellularis (nPGi), which © 2007 International Society for Sexual Medicine
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Anti-SERT Saporin Lesion of VLM and UG Reflex is located in hindbrain in the ventrolateral medulla (VLM). Lesions of the VLM release the spinal inhibition of the UG reflex, facilitate ex copula sexual reflexes, and increase sexual function in exhaustion models [7,8]. Serotonin-containing neurons and serotonin receptors are located in the VLM, in regions that overlap with sites thought to mediate this inhibition, as well as in the midline raphe [1,8,9]. In addition, serotonin neurons originating in the VLM innervate spinal circuits that regulate pelvic organ function [8,10–15]. Selective serotonin reuptake inhibitors (SSRIs) given to treat psychological disorders such as depression and anxiety often cause reduced sexual function, including decreased arousal and anorgasmia [16,17]. SSRIs act on serotonin reuptake mechanisms via the serotonin reuptake transporter (SERT) that mediates inactivation of serotonin following its release at synapses. Inhibition of the serotonin reuptake system results in increased availability of serotonin that is thought to mediate the delayed ejaculation in humans [16,17]. Studies supporting this hypothesis have demonstrated that peripheral administration of serotonin may act via 5-hydroxytryptamine (5-HT)1B and 5-HT2C receptors to inhibit ejaculatory behavior [8,16,18–21]. However, facilitation of ejaculation through 5-HT1A receptors has also been reported [18,22]. Previous studies demonstrated that intrathecal or intracerebroventicular administration of 5,7 dihydroxytryptamine (5,7 DHT) removed the descending inhibition of the UG reflex, which was associated with a significant decrease in serotonin levels in the spinal cord [23]. The origin of spinal serotonergic inputs arises primarily from neurons in the pons and medulla. In order to further understand the link between SSRIs and ejaculatory-like responses, the present study examined the effect of lesions of ventral medullary neurons containing the SERT on the UG reflex. Removal of neurons containing the SERT receptor was achieved using the ribosomeinactivating protein, saporin, which was conjugated to a monoclonal antibody to the fourth extracellular domain of the SERT (Advanced Targeting Inc.) [24]. Saporin conjugates act as targeted neurotoxins that result in selective death of neurons containing the appropriate receptors [24–27]. If sexual reflexes are influenced by saporin removal of brain neurons containing the SERT, this would further confirm a role for central serotonin in regulating ejaculatory-like reflexes. This technique has proven successful in studying the
central nervous system control of respiration and bladder function [28–30]. Methods
Anti-SERT Saporin Injections All experimental procedures involving animals were approved by the University of North Carolina Institutional Animal Care and Use Committee in accordance with National Institute of Health guidelines. Male Sprague–Dawley rats (N = 22) were anesthetized with a mixture of ketamine and xylazine (50 mg/kg + 4 mg/kg, intraperitoneally, respectively) and placed into a stereotaxic apparatus. A craniotomy was performed to allow access to the nPGi using aseptic techniques. A Hamilton syringe (1 mL) was lowered into the rostral nPGi (coordinates 2.4 mm caudal to lambda, 1.2 mm lateral to the midline, and 8.5–8.75 mm deep) and anti-SERT saporin (anti-SERT SAP; Advanced Targeting Systems, San Diego, CA, USA, 80 nL, 1 mM) was slowly injected into the brain. Four control animals received vehicle injections of saline (80–100 nL, containing 0.1% pontamine sky blue). The syringe remained in place for 5 minutes after the end of the injection then was slowly removed. Injections were made bilaterally. The craniotomy was sealed with bone wax and the skin overlying the skull was sutured. Three additional control animals received no injections. Physiology Following a recovery period of 10–18 days [24,31], all rats were anesthetized with urethane (1.2– 1.4 g/kg intraperitoneally). The carotid artery and jugular vein were cannulated for measurement of arterial pressure and administration of drugs, respectively. Rats were maintained at ~38°C with a thermostatically controlled heating blanket. A catheter (PE 50), connected to an infusion pump and a pressure transducer, was inserted into the urethra via a bladder incision and tied in place. Bipolar silver electrodes were used to obtain electromyographic (EMG) recordings from the bulbospongiosus muscles. Recording signals were fed into a high impedance preamplifier, displayed onto a polygraph and oscilloscope, and fed into an audio monitor and Computer (PowerLab 8 and Chart 5.0 from AD Instruments, Colorado Springs, CO, USA). Raw EMG signals were rectified and integrated with a 100-millisecond time constant. Infusion of saline (0.4 mL/min) into the urethra and brief occlusion of the urethral meatus was used to J Sex Med 2007;4:322–331
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evoke the UG reflex (poststimulus rhythmic contractions of the bulbospongiosus muscles) [2]. The presence or absence of the UG reflex was examined in the intact preparation. Subsequently, the spinal cord was cut at T9–10 and after at least 30 minutes the presence of the UG reflex was re-examined.
Immunohistochemistry Immediately following physiological recordings rats were perfused with 4% paraformaldehyde while under deep anesthesia. Brain tissue was removed and placed in 4% paraformaldehyde overnight and then transferred to 30% sucrose. Coronal sections (35 mm) of the brainstem were cut and transferred to cyroprotectant [32]. A series of sections (every 0.2 mm) was processed for immunohistochemical staining of tryptophan hydroxylase (TPOH, the rate-limiting enzyme involved in the synthesis of serotonin) in order to examine the number and location of serotonin-containing neurons. Separate sections (every 0.4 mm) were incubated with dopamine beta-hydroxylase (DBH) in order to examine the number and location of norepinephrine- and epinephrine-containing neurons. Free floating sections were rinsed in potassium phosphate-buffered saline and then incubated overnight with either a mouse antibody against TPOH (1:5,000; Sigma, St. Louis, MI, USA), or a mouse antibody against DBH (1:1,000; Chemicon, Temecula, CA, USA). After washing, sections were incubated in biotinylated rabbit anti-mouse immunoglobulin G (1:500; Vector Laboratories, Burlingame, CA, USA). Sections were then incubated in avidin–biotin complex (ABC Elite 1:1,000; Vector Laboratories) and reacted with diaminobenzidine solution (Vector Laboratories). All sections were mounted on plusR slides, and coverslipped. A series of sections were mounted and counterstained with thionin in order to visualize the injection sites. Data Analysis The numbers of immunostained cells in the raphe pallidus, raphe obscurus, raphe magnus, ventral surface, parapyramidal region, and the VLM (Figure 1) were counted in each section. The caudal extent of the inferior olives was designated as 0 and the section where the trapezoid bodies crossed the midline was found at ~3 mm rostral. In addition, the number of immunostained neurons in the A5, and caudal median raphe and dorsal raphe area were counted [33]. The person countJ Sex Med 2007;4:322–331
Figure 1 Diagrammatic representations of the medulla indicating the locations in which tryptophan hydroxylase (TPOH)-positive cells were counted. Cells stained overlying the pyramids (pp) were included in the raphe magnus (RM) counts more rostrally. These sections represent approximately 0.6 mm and 2.4 mm from the caudal extent of the inferior olives. Amb = nucleus ambiguus; FN = facial nucleus; IO = inferior olives; NTS = nucleus tractus solitarius; p = pyramids; pp = parapyramidal region; Rp = raphe pallidus; RM = raphe magnus; RO = raphe obscurus; Ve = vestibular nucleus; VLM = ventrolateral medulla; VS = ventral surface.
ing the cells was blinded to the treatment group. The number of immunostained cells in each area was compared between groups of animals treated with anti-SERT SAP that demonstrated the UG reflex in the spinally intact preparation and controls that did not demonstrate the UG reflex in the intact preparation. Statistical significance (P < 0.05) was determined with a one-way anova followed by Scheffe’s post hoc tests using SPSS 13.0 for Windows. Results
Physiology Three animals (one saline and two anti-SERT SAP) were excluded from the data analysis because of physical damage from the injections that was found upon subsequent histological examination. In spinally intact controls (saline-injected and noninjected) the UG reflex could not be evoked (Figure 2, control, intact). However, after cutting the spinal cord, rhythmic contractions of the bulbospongiosus muscles were reliably evoked at low thresholds (25–40 mm Hg) in response to urethral distension (Figure 2, control, spinalized).
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Figure 2 Effect of VLM injections of anti-SERT SAP on the UG reflex. Saline (control) or anti-SERT SAP was injected bilaterally into the VLM 14 days before recording the UG reflex. In controls with intact spinal cord the UG reflex could not be evoked. Pretreatment with anti-SERT SAP exposed the UG reflex in the intact preparation (anti-SERT SAP, intact). After spinal cord transection at T10 (spinalized) the UG reflex was present in both the control and antiSERT SAP animals. The UG reflex can be seen as rhythmic firing of the bulbospongiosus muscles (BS EMG) following release of the occlusion (black lines at bottom of urethral pressure trace show the occlusion period). BS EMG was rectified and integrated with a 200-millisecond time constant.
In contrast, the UG reflex was present in eight animals (62%) that received anti-SERT SAP injections before the spinal cord was transected (Figure 2, anti-SERT SAP, intact). In these animals the UG reflex was consistently evoked at low thresholds (25–55 mm Hg) and the latency to initiation of the UG reflex was 41 ⫾ 6.9 seconds (range 10–65 seconds) (Figure 2, anti-SERT SAP, intact). The latency to the UG reflex after spinalization was significantly shorter than before spinal cuts in both the control and anti-SERT SAP groups (Figure 2, anti-SERT SAP, spinalized). The remaining animals (38%) that received antiSERT SAP showed either inconsistent responses or no rhythmic contractions of the bulbospongiosus muscles prior to spinal cord transection.
Immunohistochemistry TPOH Staining In the medulla the majority of TPOH-positive cells were found in the raphe obscurus and raphe magnus at 0–2 mm, and 2–3 mm, respectively (Figure 3). TPOH-containing cells in the raphe pallidus were located primarily 0.2–2.2 mm at the level of the inferior olives (Figure 3). TPOHpositive cells in the VLM included cells lateral to the pyramidal tract, on the ventral surface of the medulla, and in the parapyramidal region (cells lying over the pyramids in the gigantocellular reticular formation), as shown in Figure 1. In the VLM the majority of cells staining for TPOH were found between 1.6 and 2.8 mm; this area
overlaps with the rostral nPGi [33]. At 2–3 mm the parapyramidal cells formed an extension of the raphe magnus and thus TPOH-containing cells at these levels were included in the raphe magnus counts. This area consists of the B3 region of the serotonergic neurons [34]. TPOH-positive cells were also found in the median raphe and dorsal raphe area. Cell counts in animals that received anti-SERT SAP and displayed UG reflexes in the spinally intact preparation were compared with controls. Presence of the UG reflex in the intact preparation was associated with a significant decrease in TPOH-positive cells in the raphe magnus, raphe obscurus, raphe pallidus, and VLM (Figures 3 and 4). A reduction in the number of TPOH-positive cells was found throughout the length of the medulla in the raphe obscurus and raphe magnus. In the VLM a significant decrease in the number of TPOH-containing cells was also found, particularly from 2.0 to 3.2 mm at the level of and rostral to the injection site (Figures 3 and 4). However, there was no difference between groups in the numbers of TPOH cells counted on the ventral surface, in the parapyramidal region, the median raphe or dorsal raphe (Figure 5). Animals that received anti-SERT SAP and showed either inconsistent or no rhythmic contractions of the bulbospongiosus muscles prior to spinal cord transection had variable cells counts that were on average not different from controls. J Sex Med 2007;4:322–331
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Figure 3 Graphs illustrating the rostrocaudal distribution of tryptophan hydroxylase (TPOH)-positive cells in the raphe pallidus, raphe obscurus + raphe magnus, ventrolateral medulla, and total cell counts. Each data point is the average of the total number of cells counted at 0.2 mm intervals (the bars show the SE of the mean). Controls (solid lines, filled circles) and antiSERT saporin group (dashed lines, open circles) that displayed the UG reflex in the spinally intact preparation are plotted. N = 4–7.
DBH Staining Dopamine beta-hydroxylase-containing neurons were found in the VLM and in the A5 region. In the caudal medulla (0–1.8 mm) an average of 39 ⫾ 12 and 34 ⫾ 11 DBH-positive cells per section were found in the control and anti-SERT SAP groups, respectively. In the A5 region 32 ⫾ 10 and 27 ⫾ 11 DBH-positive cells per
Figure 4 Histogram illustrating the total number of tryptophan hydroxylase (TPOH)-positive cells counted in the medulla in controls (open bars) and the anti-SERT SAP group that displayed the UG reflex in the spinally intact preparation (solid bars). RO = raphe obscurus; RM = raphe magnus; RP = raphe pallidus; VLM = ventrolateral medulla; VS = ventral surface; PP = parapyramidal region; MR = median raphe; DR = dorsal raphe. N = 5–7. *P < 0.05, **P < 0.001 indicate a significant difference between the control and anti-SERT SAP group.
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section were found in the control and anti-SERT SAP groups, respectively. There were no significant differences between the groups.
Discussion
Partial reduction of serotonin containing neurons in the medullary raphe and VLM allowed the elicitation of the UG reflex in the anesthetized spinally intact preparation. The associated decrease in TPOH staining in the VLM with the appearance of perineal muscle activity is consistent with previous studies that suggested that descending serotonergic inputs from the nPGi to the lumbosacral spinal cord are involved in mediating the supraspinal inhibition of ejaculatory-like responses [35–37]. Lesions of the ventral medulla in the region of the nPGi removed the supraspinal inhibition of the UG reflex, facilitated ex copula penile reflexes, and improved sexual behavior in sexually exhausted rats. Stimulation of the nPGi also alters pudendal motor output and produces inhibition of penile reflexes, and neurons in this region receive convergent information from multiple areas including the penis and UG tracts [38–40]. Serotonergic neurons in the VLM project directly to pudendal motor neurons and preganglionic neurons that are activated in response to UG stimulation [7,14,15,41–46]. Taken together these data indicate that brainstem-spinal serotoner-
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Figure 5 Photomicrographs showing tryptophan hydroxylase (TPOH) neurons in the ventrolateral medulla (A and B, at 2 mm level) and raphe magnus (C and D, at 2.6 mm level) in control (A and C) and anti-SERT saporin injected (B and D) animals. Note: more TPOH-labeled cells in the controls. Scale bar = 60 mm.
gic pathways participate in the regulation of ejaculatory-like responses including the UG reflex. The number of TPOH-containing neurons in the anti-SERT SAP group was also reduced in the medullary raphe. Previous studies have suggested that the raphe obscurus, raphe pallidus, and dorsal raphe may be involved in sexual function [47]. Lesions of the raphe obscurus decreased the number of intromissions and ejaculations [47]. However, the occurrence of penile reflexes was not affected by raphe obscurus lesions [42]. Neurons containing the SERT are also present in the median raphe and dorsal raphe and the dorsal raphe is sensitive to gonadal steroid hormone treatment, suggesting this nucleus may also be involved in sexual function. In one study, large injections of 5,7 DHT into the dorsal raphe decreased ejaculatory latency in awake sexual behavior tests [41]. However, no changes in the numbers of TPOH neurons were found in the median raphe and dorsal raphe in the present study suggesting that these areas are not essential for the supraspinal inhibition of the UG reflex. Very few studies have examined brainstem changes in neural activity that is associated with sexual behavior. One study reported significant changes in the number of c-fos-activated neurons in the VLM and raphe pallidus after ejaculation
that was thought to be related to the postejaculatory refractory period [43]. Because of the close proximity of the medullary raphe and the VLM and their known reciprocal projections, lesions of or injections into one site may affect the function of the other [48–51]. In addition, SERT is found primarily on axons, dendrites, and terminals of serotonin neurons [52], therefore loss of serotonergic raphe neurons in the present study may have occurred through raphe projections to the VLM. No decrease in TPOH-positive cell numbers on the ventral surface and caudal parapyramidal regions was found in the present study suggesting that these serotonergic neurons are not involved in tonic inhibition of the UG reflex. Many but not all the spinally projecting neurons in the ventral medulla contain serotonin [8–10,53,54]. In addition, other neurotransmitters innervate the lumbosacral spinal cord. In the present study no change in the number of DBHcontaining neurons was found suggesting that the saporin injections did not affect norepinephrine and epinephrine neurons. Other neurotransmitters, for example, substance P and thyrotropinreleasing hormone containing neurons, also project to the spinal cord and these neurotransmitters are colocalized within a distinct proportion of serotonergic neurons [9,54–56]. Therefore, destruction of neurons containing the SERT may J Sex Med 2007;4:322–331
328 also affect other neurotransmitters colocalized in these neurons. Further studies are required to investigate the role of these multiple neurotransmitters in the control of sexual function. Examination of spontaneous, rather than reflex-induced, erectile and ejaculatory responses in rats has been conducted using pchloroamphetamine (PCA), which results in the release catecholamines and serotonin from neurons [57]. These studies demonstrated spontaneous ejaculatory-like responses after systemic administration of PCA in both awake and anesthetized models [58–61], suggesting that release of monoamines can facilitate or induce spontaneous ejections and ejaculation. While PCA can act in the brain to produce its effects on sexual behavior, evidence for a spinal site of action has also been provided [57,58,62–64]. Systemic administration of PCA also acts on peripheral organs and nerves and a recent study demonstrated that ejaculatory responses induced by PCA require a peripheral-spinal pathway [65]. While it is generally thought that the PCA effects on sexual reflexes are via the serotonin system, PCA results in an increase in catecholamines, including dopamine, which has a potent effect on facilitating sexual responses [66,67]. In addition, activation of 5-HT1A receptors facilitate ejaculation while activation of 5-HT1B/2C inhibit sexual responses, thus the resultant effect on ejaculatory reflexes may depend on the receptors predominately activated [18–20]. Therefore, multiple serotonin pathways (e.g., brain, brain-spinal, and peripheral-spinal), receptors, and mechanisms (inhibitory and facilitatory) are involved in regulating erectile and ejaculatory events. Previous studies have successfully used saporin conjugates, including anti-SERT SAP, to selectively destroy neurotransmitter-specific cells in the brain [24,28–30,68]. The present study concurs with these studies in that saporin conjugates can selectively destroy specific neurotransmitter populations. However, further studies are required to investigate the effect on neurons containing multiple neurotransmitters.
Acknowledgments
The authors would like to thank Dr. Rong-S Cai for his expert technical assistance. Corresponding Author: Lesley Marson, PhD, The University of North Carolina, Division of Urology, Department of Surgery, 4024 Burnett Womack BuildJ Sex Med 2007;4:322–331
Gravitt and Marson ing, Chapel Hill, NC 27599, USA. Tel: (919) 966-9252; Fax: (919) 966-0098; E-mail: [email protected] Conflict of Interest: None declared. References
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In the last decade, efforts have been made to investigate the neurobiology of ejaculation and in parallel pharmacological management of premature ejaculation (PE) has been proposed. Selective serotonin reuptake inhibitors (SSRIs) used to treat depression have long been known to cause sexual dysfunctions, notably delayed ejaculation and sometimes anejaculation. Based on this
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Anti-SERT Saporin Lesion of VLM and UG Reflex observation, numerous clinical studies have investigated the potential interest of chronic antidepressant SSRIs in lengthening ejaculation time in PE patients [1] and, as recently reported, on demand treatment with the short-acting SSRI dapoxetine appears effective and safe [2]. However, the mechanism of action by which SSRIs delay ejaculation is still not fully understood [3]. The present study is a contributive effort made to clarify this issue. Using the urethrogenital reflex model in anesthetized rats coupled with a highly specific neutotoxic lesioning approach, the authors showed the primary role of serotonergic neurons expressing serotonin transporter and located in the ventrolateral medulla and medullary raphe on the control of the ejaculatory motor reflex. Although the experimental paradigm used here is extra physiological and mimics only some aspects of the ejaculatory response, it has been previously shown useful in improving our understanding on the control of perineal striated muscles activity, a key element in the expulsion phase of ejaculation. Nonselective electrolytic lesion of the paragigantocellular nucleus, which includes ventrolateral medulla, has been shown to reverse fluoxetine-delaying effect on ejaculation in male rats [4], tending to give a physiological meaning for the present findings. However, this remains to be confirmed in behavioral investigations using the elegant and selective approach described here. Finally, it is to
be noticed that the present results provide indications for the differentiation of the mechanism of action between antidepressant and ejaculationretarding properties of SSRIs. François Giuliano, MD, PhD Department of Neuro-Uro-Andrology Physical Medicine and Rehabilitation, Raymond Poincare Hospital, Garches France E-mail: [email protected]
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