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Research Report

Role of the lateral preoptic area and the bed nucleus of striaterminalis in the regulation of penile erection

Hiroshi Iwasakia,b, Eiichi Jodoa, Akihiro Kawauchic, Tsuneharu Mikic,Yukihiko Kayamaa, Yoshimasa Koyamad,⁎aDepartment of Neurophysiology, Fukushima Medical University, 1 Hikari-ga-oka, Fukushima 960-1295, JapanbDepartment of Urology, Maizuru Kyosai Hospital, 1035, Hama, Maizuru, Kyoto 625-0037, JapancDepartment of Urology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, JapandDepartment of Science and Technology, Fukushima University, 1-Kanaya-gawa, Fukushima 960-1296, Japan

A R T I C L E I N F O

⁎ Corresponding author. Fax: +81 24 548 8440.E-mail address: koyamay@sss.fukushimaAbbreviations: BS, bulbospongiosus; BST

electroencephalogram; EMG, electromyogramlateral septum; MPOA, medial preoptic areaREM, rapid eye movement

0006-8993/$ – see front matter © 2010 Elsevidoi:10.1016/j.brainres.2010.08.006

A B S T R A C T

Article history:Accepted 3 August 2010Available online 10 August 2010

To elucidate the role of the preoptic area (POA) in the regulation of penile erection, weexamined the effects of electrical stimulation in and around the POA on penile erection in rats,which was assessed by changes in pressure in the corpus spongiosum of the penis (CSP) andelectromyography (EMG) of the bulbospongiosus (BS) muscle. In unanesthetized andanesthetized rats, four types of responses were induced by stimulation in and around thePOA; (1) normal type responses, which were similar to spontaneously occurring erections,characterized by slow increase in CSP pressure and sharp peaks concurrent with BS musclebursting; (2) muscular type responses, which included sharp CSP pressure peaks (muscularcomponent) with almost no vascular component; (3) mixed type responses, which included asequence of high-frequency CSP peaks followed by low-frequency CSP peaks; and(4) micturition type responses, which had higher-frequency and lower-amplitude CSP peaksthan other responses which were identical to those of normal micturition. In unanesthetizedcondition, erections were evoked by stimulation of the lateral preoptic area (LPOA), medialpreoptic area (MPOA), bed nucleus of the stria terminalis (BST), paraventricular nucleus (PVN),reuniens thalamic nucleus (Re) and lateral septum (LS). Lower-intensity stimulation evokederections from the LPOA, BST, PVN and RE, but not the MPOA. In anesthetized condition,stronger stimuli were required and effective sites were restricted to the LPOA, MPOA and BST.These findings suggest that the lateral and medial subdivisions of the preoptic area playdifferent roles in mediating penile erection.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Penile erectionMedial preoptic areaLateral preoptic areaBed nucleus of the stria terminalisParaventricular nucleus

-u.ac.jp (Y. Koyama)., bed nucleus of the stria terminalis; CSP, corpus spongiosum of the penis; EEG,; FB, flaccid baseline; LDT, laterodorsal tegmental nucleus; LPOA, lateral preoptic area; LS,

; NCE, non-contact erection; PVN, paraventricular nucleus; Re, reuniens thalamic nucleus;

er B.V. All rights reserved.

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1. Introduction

It has been reported that the medial part of the preoptic area(POA) and the structures surrounding it play critical roles insexual function (Coolen, 2005). Electrical stimulation of themedial preoptic area (MPOA) evoked sexual behavior (Malsbury,1971; Merari and Ginton, 1975) or penile erection (Giuliano et al.,1996, 1997; Sato and Christ, 2000), while lesions of the MPOAcaused severe deficits in copulation (Ginton and Merari, 1977;Hansen et al., 1982). Single neuronal activity in the MPOAexhibited a close correlation with male sexual behavior in ratsand monkeys (Oomura et al., 1983; Shimura et al., 1994).Activation of the paraventricular nucleus (PVN) by NMDAinjection caused penile erection (Melis et al., 1997). Lesions ofthe bed nucleus of the stria terminalis (BST) induced deficiencyin some aspects of sexual behavior (Valcourt and Sachs, 1979;Liu et al., 1997a). Schmidt et al. (2000)have reported that ibotenicacid lesions of the lateral preoptic area (LPOA) disrupted penileerection during rapid eye movement (REM) sleep, while leavingwaking-state erections intact. These reports suggest that thereare two regulatory systems for penile erection located in thePOA; the MPOA system, which is involved in sexual behavior-relatederection,and theLPOAsystem,which is crucial for sleep-related erection.

To test this hypothesis, we examined the effects ofelectrical stimulation in and around the POA on penileerection in rats. For this experiment, we used fine carbonfiber electrodes which are movable and able to systematicallystimulate numerous points in one animal, and measurederectile tissue pressure using a telemetric monitoring tech-nique (Nout et al., 2007; Schmidt et al, 1994).

2. Results

2.1. Responses in unanesthetized condition

Stimulation at 925 sites in and around the preoptic areayielded four different types of responses; normal, musculartype, mixed type and micturition type. Fig. 1 shows an

Fig. 1 – Normal response after stimulation (100 μA) of the lateralto that of a spontaneous erection and BS muscle activity synchrocorpus spongiosum of the penis; EMG, electromyogram; EEG, ele

example of a normal response evoked by delivery of astimulus of 100 μA to the lateral preoptic area (LPOA). Thisstimulus induced, with a latency of 20 seconds, a slowincrease in CSP pressure reaching 30 mmHg and sharp CSPpressure peaks riding on the slow CSP pressure increase. TheBS muscle exhibited bursting discharges in accordance withthe CSP pressure peaks. This pattern of erection was similar tothat of spontaneous erections. Fig. 2 shows the profile of theresponses obtained at different depths of a track passingthrough the LPOA. At a depth of 7.0 mm from the surface,erection was evoked by stimulation with 100 μA (Trace A).Erection was not evoked by the same stimulus at a point 0.4-mm deeper (Trace B). Although small BS muscle activity wasevoked at depths of 7.8 mm and 8.2 mm, no erections wereevoked (Traces C and D, respectively). At 8.6 mm from thesurface, a 50-μA stimulus evoked erection (Trace F). However,with a stimulus of 50 μA, no response was observed at points0.2 mm away (dorsal and ventral) from the effective point(Traces E and G). These findings indicate that the effects of a50-μA stimulus do not spread further than 0.2 mm, whilethose of a 100-μA stimulus are limited to 0.4 mm from thestimulation point.

As shown in Fig. 3, the muscular type response, similar tonormal responses, exhibited a series of sharp CSP pressurepeakswith a characteristically small vascular component (lessthan 30 mmHg). The sharp CSP pressure peaks occurredsimultaneously with BS muscle activity (they are not shownin Fig. 3, since in this preparation the recording wires for BSmuscle were removed). The sharp CSP pressure peaksappeared more regularly than those in normal responses.The coefficient of variation of the peak interval in musculartype responses, including those in both unanesthetized andanesthetized rats, was significantly smaller than that fornormal responses (p<0.01 by Mann–Whitney U test).

Fig. 4 shows an example of amixed type response in which,in the vascular component, two types of CSP pressure peaksoccur sequentially; initial high-frequency peaks (phase A)followed by low-frequency peaks (phase B). As Table 1indicates, the peak frequencies of phase A were significantlyhigher than those in normal responses (p<0.001) and muscu-lar type responses (p<0.001), while the peak frequencies of

preoptic area, which caused a CSP pressure pattern similarnous with CSP pressure peaks. BS, bulbospongiosus; CSP,ctroencephalogram.

Fig. 2 – Depth profiles of effects of electrical stimulation atdepths of 7.0 mm (A) to 8.8 mm (G) from the brain surface.Circles, effective points; crosses, ineffective points; stim,stimulation.

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phase B were within the range of normal responses andmuscular type responses. In addition, the peak-to-peakduration of phase A was significantly shorter than those ofnormal type (p<0.05) and muscular type (p<0.05) responses.

The response shown in Fig. 5 consisted of regular, high-frequency, low-amplitude CSP peaks. The peak frequency wasfrom 9.7 to 10.3 Hz, and within the range observed in ourprevious study (Yamao et al., 2001) or close to the dischargefrequency of the sphincter muscle discharge during naturallyoccurring micturition (Nout et al., 2007). This response wastherefore termed micturition type response. The peak fre-quency of the micturition type response was the highest of allresponses (p<0.001), and the high-frequency peaks weresynchronous with BS muscle bursts. In micturition typeresponses, the maximum peak amplitudes were smaller

Fig. 3 – Muscular type response after stimulation (300 μA) of thealmost no vascular component but accompanied by steep periodiwas deleted from the figure.

than those of normal, muscular type and mixed typeresponses (phase B) (p<0.05). Of 67 responses obtained inunanesthetized condition, 51 (76.1%) were normal, 8 (11.9%)were mixed type, 6 (9.0%) were muscular type and 2 (3.0%)were micturition type (Table 1).

Fig. 6A indicates the sites from which positive responseswere evoked on stimulation in unanesthetized animals. Of 364sites in and around the preoptic and hypothalamic areas,responses were evoked from 39 sites including the lateralpreoptic area (LPOA), medial preoptic area (MPOA), bednucleus of the stria terminalis (BST) and paraventricularnucleus (PVN). In addition, stimulation in surrounding areasincluding the reuniens thalamic nucleus (Re) and the lateralseptum (LS) was also effective. Stimulation in the thalamus,anterior hypothalamus and lateral hypothalamus was noteffective. The ratios of effective sites to stimulation sites in theLPOA, MPOA and BST were 13/131 (9.9%), 7/55 (12.7%) and 8/94(8.5%), respectively, and were highest in the PVN (4/10; 40%)(Table 2). As shown by large circles indicating stimulusintensity of less than 50 μA, erections were more effectivelyevoked from the BST at the level of Br 0.26 to Br 0.40, posteriorhalf of the LPOA (Br 0.92 to Br 1.30) and the PVN (Br 1.30). On theother hand, in the case of responses obtained from the MPOA,stimulus intensities above 100 μA (in most cases, 200 μA or300 μA) were required.

2.2. Responses in anesthetized condition

Under urethane anesthesia, four types of responses – normal,muscular type,mixed typeandmicturition type –wereobserved(Table 1). Of a total of 32 responses, 17 (53.1%) were normal, 2(6.3%) were mixed type, 8 (25.0%) were muscular type and 5(15.6%) were micturition type. Compared with unanesthetizedcondition, the proportion of muscular type responses wassignificantly higher (p<0.02 by χ2 test.) In anesthetized condi-tion, effective sites were restricted to the LPOA, MPOA and BST(Fig. 6B, Table 2). The ratios of effective sites to stimulation sitesin the LPOA, MPOA and BSTwere 8/170 (4.7%), 13/200 (6.5%) and4/78 (5.0%), respectively. The ratios were lower than those inunanesthetized condition (13/131 (10%), 7/55 (12.7%) and 8/94(8.5%), respectively). In total, responsiveness under urethaneanesthesia (25/561, 4.5%) was significantly lower than inunanesthetized condition (39/364, 10.7%) (Table 2). Strongerstimuli (more than 100 μA) were required to elicit erection than

paraventricular nucleus, with a CSP pressure pattern withc CSP peaks. Since the BS EMG could not be recorded, the trace

Fig. 4 – Mixed type response after stimulation (50 μA) of the lateral preoptic area, which is composed of a CSP pressurepattern with high-frequency CSP peaks (Phase A) followed by low-frequency CSP peaks (Phase B).

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in the unanesthetized condition. Compared with unanesthe-tized condition, latencies were longer (p<0.01), the frequencyand amplitude of CSP pressure peaks were lower (p<0.001,p<0.01, respectively), and the duration of peaks was longer(p<0.001). Under anesthesia, the frequencies of normal re-sponse (0.02–0.52, 0.25 Hz)were significantly lower than thoseofmuscular type responses (0.31–1.33, 0.62 Hz) (p<0.01) (Table 1).

3. Discussion

In rats in unanesthetized and anesthetized conditions, fourtypes of responses – normal, muscular type, mixed type, andmicturition type – were induced by stimulation in and aroundthe preoptic area. Normal responses were most similar tospontaneously occurring erections. Normal responses werecomposed of a slow increase in CSP pressure and sharp peaksconcurrent with BS muscle bursting. The amplitude, frequen-cy and duration of these responses were within the rangesobserved in our previous studies for spontaneous erections ornormal erections (Gulia et al., 2008; Salas et al., 2007). Theevoked response was thus quite similar to physiologicalerection. Induction of muscular type responses, with sharpCSP pressure peaks (muscular component) with almost novascular component, suggests that in the preoptic area the

Table 1 – Parameters of four types of stimulus-evoked response

*p<0.05; **p<0.01; ***p<0.001 by Mann–Whitney U test with correction.

neural system regulating themuscular component is separatefrom that regulating the vascular component. Electricalstimulation often induced slow CSP pressure increase (vascu-lar component) without, or with only a single, sharp CSP peak(unpublished observation), suggesting that the neural systemregulating the vascular component was selectively activated.Induction of mixed type responses, which included a se-quence of high-frequency CSP peaks followed by low-frequen-cy CSP peaks, suggested that two systemswere activated, one,generating high-frequency BS muscle activity and the othergenerating BS muscle activity similar to that observed innormal responses or spontaneous erections. Micturition typeresponses had higher-frequency and lower-amplitude CSPpeaks than other responses. The peak frequency (5.16–10.29 Hz) covered the range of the frequency of the externalsphincter muscle discharges during micturition (Kruse et al.,1990; Yamao et al., 2001). In some cases, we observed voidingwhen the high-frequency CSP fluctuation occurred (unpub-lished observation). Micturition type responses thus appear toreflect normal physiological micturition. The micturitioncenter in the brainstem (Barrington's nucleus) receivesprojection from the preoptic area (Rizvi et al., 1994). Thestimuli that induced micturition type responses were thustransmitted to Barrington's nucleus through the preoptic-Barrington's nucleus pathway.

s.

Fig. 5 – Micturition type response, evoked by stimulation (50 μA) of the lateral septum and composed of high-frequency CSPpressure peaks without a vascular component.

Fig. 6 – Coronal sections showing the locationof effective sites fromwhicheach typeof responsewasevoked. (A) Unanesthetizedcondition; (B) anesthetized condition. Red circles, normal responses; yellowcircles,muscular type responses; blue circles,mixedtype responses; green circles, micturition type responses; circles with multiple colors, sites from which two or three types ofresponses were evoked; large circles, responses evoked by less than 50 μA stimulation; medium-sized diamonds, responsesevoked by less than 100 μA stimulation; small circles, responses evoked by less than 300 μA stimulation. AC, anteriorcommissure; BST, bednucleusof the stria terminalis; LPOA, lateral preoptic area; LS, lateral septum;MPOA,medial preoptic area;OX, optic chiasm; PVN, paraventricular nucleus; Re, reuniens thalamic nucleus; SM, stria medullaris.

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Table 2 – Ratio of effective sites to total stimulation sites ineach anatomical area.

Stimulationsites

Positive sites/total sites

Unanesthetized Anesthetized

LPOA 13/131 (0.099) 8/170 (0.047)MPOA 7/55 (0.127) 13/200 (0.065)BST 8/94 (0.085) 4/78 (0.05)PVN 4/10 (0.4) 0/13 (0)RE 3/11 (0.272) 0/9 (0)LS 4/21 (0.19) 0/10 (0)TH 0/33 (0) 0/60 (0)AH 0/4 (0) 0/17 (0)LH 0/5 (0) 0/4 (0)Total 39/364 (0.107) 25/561 (0.045)

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In unanesthetized condition, the proportions of normal,muscular type, mixed type and micturition type responseswere 76.1% (51/67), 9.0% (6/67), 11.9% (8/67) and 3.0% (2/67),respectively. The proportion of normal type was higher whilethat of micturition type was lower than in a brainstem(mesopontine tegmentum) stimulation study (Salas et al.,2007), in which the proportion of normal responses was 39.7%(81/204) and that of micturition type responses 33.8% (69/204).This is probably because themesopontine tegmentum ismoreclosely related to micturition than the preoptic area. Thevalues obtained in the present study were similar to thoseobtained in the septum stimulation study, in which thefrequency of normal responses was higher (89.0%; 65/73)than those of other responses (mixed type [6.8%; 5/73] andmicturition type [4.1%; 3/73]) (Gulia et al., 2008).

Under anesthetized condition, compared with unanesthe-tized condition, latencies were longer (p<0.01), the frequencyand amplitude of CSP pressure peaks were lower (p<0.001,p<0.01, respectively) and peak-to-peak duration was longer(p<0.001). It has been reported that NMDA receptors have afacilitatory role in penile erection (Aioun and Rampin, 2006;Song and Rajasekaran, 2004; Succu et al., 2006), and thaturethane suppresses the function of NMDA receptor (Hara andHarris, 2002). Suppression of NMDA receptors by urethanewould thus have caused longer latencies and lower peakfrequencies. It is also possible that, in normal spontaneouserections, inhibitory mechanisms are at work to terminateerection. Urethane anesthesia would have suppressed suchtermination signals due to such mechanisms, resulting inlonger peak-to-peak duration. Under anesthetized condition,muscular type responses were observed frequently, indicatingthat urethane anesthesia more strongly suppressed thesystem activating the vascular component. Under unanesthe-tized condition, erections were evoked from the LPOA, MPOA,BST, PVN, Re and LS, while under anesthetized condition,stronger stimuli were required, and effective sites wererestricted to the LPOA, MPOA and BST. Under urethaneanesthesia, erection was thus evoked only from the crucialareas for regulating erection.

Schmidt et al. reported that ibotenic acid lesions of LPOAdisrupted penile erection during REM sleep, while leavingwaking-condition erection intact, and suggested that the LPOAplays a crucial role in penile erection during REM sleep (Schmidtet al., 2000). Our finding that penile erections were evoked by

stimulation of the LPOA further confirms that the neuralsubstrate for penile erection is located in the LPOA. Schmidtet al. (2001) have also reported that injections of carbachol inthe LPOA induce penile erections.We found that stimulation ofthe laterodorsal tegmental nucleus (LDT, one of the brainstemcholinergic nuclei) induced erection (Salas et al., 2007), andsuggested that the LDT is a cholinergic source for induction oferection in the LPOA.

TheMPOA is known to play crucial roles in the regulation ofreproductive function (Coolen, 2005; Dominguez and Hull,2005). Stimulation of the MPOA induces copulatory behavior(Malsbury, 1971; Merari and Ginton, 1975) and penile erection(Giuliano et al., 1996, 1997; Sato and Christ, 2000). However,several studies have not supported a role for the MPOA inpenile erection. Schmidt et al. (2000) found that MPOA lesionshad almost no effect on waking-state or REM sleep-relatederection. Liu et al., (1997a) have reported that male rats withMPOA lesions exhibit severe deficits in copulation but little orno decrement in non-contact erection (NCE), while BST lesionscause impairment of NCE with only slight deficits in copu-lation, suggesting that the BST plays an important role inmediating NCE. In the present study, although erections wereevoked from the MPOA, the stimulus intensities required forthem were higher than those for elicitation of erection fromthe LPOA or BST. The MPOA thus appears to play a role inpenile erection different from those of the LPOA and BST.Considering the spread of stimulus current (Fig. 2), it ispossible that the stimulation of higher intensity applied tothe MPOA invaded the LPOA or BST.

The neurons in the BST send projections to the LPOA andMPOA (Dong and Swanson, 2006). Liu et al. (1997a) havesuggested that an extra-MPOA pathway mediates NCE. Thesystem extending from BST to LPOA might correspond to thispathway.

Penile erection is evoked by the injection of apomorphine,oxytocin or glutamate in the PVN (Argiolas and Melis, 1995;Chen and Chang, 2003; Kita et al., 2006; Melis et al., 1987), whilelesions of PVN disrupts NCE (Liu et al., 1997b; Melis et al., 2001).The PVN projects directly to the intermediolateral region ofthe spinal cord (Ranson et al., 1998). In the present study,erectionwas evoked by PVN stimulationwith lower intensitiesthan the MPOA (Fig. 6A) and higher probability than any otherbrain areas (Table 2). These facts support the idea that PVNneurons receiving dopaminergic or glutamatergic inputs andprojecting directly to the spinal cord act as the final output ofthe erection regulating system in the hypothalamus.

4. Conclusion

In unanesthetized rats, penile erection was evoked by theelectrical stimulation of the LPOA, MPOA, BST, PVN RE and LS.Among them, lower-intensity stimulus was effective in theLPOA, BST, PVN and RE, but not in the MPOA. In anesthetizedcondition, the effective areas were restricted to the LPOA,MPOA and BST. The present results suggest the difference ofthe lateral and medial subdivisions of the POA, emphasizingthe role of the lateral subdivisions (LPOA and BST), in theregulation of penile erection.

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5. Experimental procedures

5.1. Animals

The study was conducted in 20 adult male Sprague–Dawley rats(350–450 g) obtained from Japan SLC Inc. and maintained on a12:12-h light/dark cycle at anambient temperatureof 22.0±0.1 °Cwith food and water available ad libitum. Of them, 13 were usedunder urethane anesthesia, while 7 were used unanesthetizedin head-restrained condition. All procedures were carriedout under the control of the Animal Research Committee inaccordance with the Guidelines for Animal Experiments ofFukushima Medical University and the Japanese Animal Protec-tion and Management Law (No. 105). All efforts were made tominimize the number of animals used and their suffering.

5.1.1. Surgical procedureFor acute experiments, animals were anesthetized with ure-thane (1.2 g/ kg, i.p.). For surgery for chronic experiments,sodium pentobarbital (50 mg/kg) was intraperitoneally injectedand additional doses were given to maintain the level ofanesthesia. As previously described, the pressure of the corpusspongiosum of the penis (CSP) was measured telemetrically(Gulia et al., 2008; Nout et al., 2007; Schmidt et al., 1994; Salaset al., 2007). In brief, the distal portion of the bulb of the corpusspongiosum of the penis (CSP) was gently exposed and thecatheter tip of the telemetric transducer (TA11PA-C40, DataSciences International, St. Paul MN) was inserted into the bulbthrough a slitmade by a needle. The body of the transducer wasfixed subcutaneously to the abdominal muscle. The catheterwas secured using a biological glue (Vetbond, 3 M Animal CareProducts) at the point of entrance of the catheter and wassutured to the fascia overlying the shaft of the penis. To recordthe electromyogram (EMG) of the bulbospongiosus (BS) muscle,a pair of stainless wires were inserted into the BS muscle andpassed subcutaneously to an incision of the skin over the skullwhichhad beenmade in advance of the implantation. Toassessvigilance state, stainless steel screws (tip diameter=1.4 mm) forrecording of the cortical electroencephalogram (EEG) wereimplanted in the skull overlying the frontal andparietal corticesand, in unanesthetized, head-restrained rats, wire electrodeswere implanted in the neck muscle to record the electromyo-gram (EMG). For painless fixation of the unanesthetized rats to astereotaxic frame during experiments, a U-shaped plastic plate(width 20mm, length 30mm, thickness 5 mm) was attached tothe skull using dental acrylic cement. Gentamicin ointment(0.1%) was applied at the sites of incision, and penicillin (60 mg)was subcutaneously injected for 4 or 5 days. The ratswere givena 1-week period for recovery.

5.2. Experimental procedures

One day before the experiment, rats were attached to thestereotaxic frame under ketamine anesthesia (50 mg/kg) usingthe U-shaped plate, and a small hole was made through theoccipital bone using a dental drill to expose the surface of thecerebral cortex above the preoptic area. The rats were deprivedof sleep for 12 h in a slowly rotating wheel (diameter 37mm,width 10 cm, 1.2 rev/min)with free access to food andwater. On

the day of the experiment, rats were returned to their cage torest for 2–3 h. They were then fixed to the stereotaxic frameusing the U-shaped plate. The stimulation electrode consistedof a glass pipette which had a carbon fiber (diameter, <10 μm;resistance, 4–6 MΩ) protruding from the tip about 20–30 μmanda cavity filled with Woods metal (Takakusaki et al., 1989). Todetermine sites most effective for induction of erection,stimulationwas applied, usually at 0.2 mm intervals, bymovingthe electrode with an oil microdrive manipulator. Stimuli weregiven using an electronic stimulator (SEN7203, Nihon Kohden,Japan)with 0.3-ms rectangular pulses of various intensities (10–300 μA) repeated at 50 Hz for 3 s. In unanesthetized condition,stronger stimuli often caused body or legmovement. Therefore,in most cases the stimuli were limited to less than 100 μA. Themost effective site along the track wasmarked by passing 20 μApositive current (DC) for 30 s.

After completion of the experiment, the rats were deeplyanesthetized with pentobarbital and perfused transcardiallywith 300 ml of physiological saline followed by 300 ml of 10%formalin. The brain was then removed, postfixed in the samefixative, soaked in 30% sucrose and sectioned in the coronalplane at a thickness of 50 μm. The sections were stained withneutral red. Stimulation sites were identified under lightmicroscopy using standard nomenclature according to the ratbrain atlas of Paxinos and Watson (1997).

5.2.1. Definition of responsesAs shown in Fig. 1, erection is composed of a slow CSP pressureincreaseandsharpCSPpressurepeaks concurrentwithburstingBS muscle activity. The slow CSP pressure increase reflects theflow of blood into the CSP caused by the distention of the bloodvessels supplying the CSP, and is termed the vascular compo-nent, while the sharp peaks reflect each of the erectile events ofthe penis (flip), which are caused by bursting discharges of theBS muscles (muscular component). In the rat, the vascularcomponent is associated with an increase in the baselineerectile tissue pressure from approximately 10–15mmHg inthe flaccid state (Flaccid Baseline, FB) to a tumescence pressureup to approximately 50–70mmHg. The muscular component iseasily identified by the sharp, suprasystolic CSP pressure peaksoccurring on top of the tumescence pressure. Normal erectionwas defined as a tumescence pressure at least 30mmHg higherthan theFB.Thestart oferectionwasdefinedas the time theCSPpressure reached FB+30 mmHg, while the end of erection wasdefined as the time when CSP pressure fell below FB+30mmHgfor more than 15 s. To distinguish between two erectionsoccurring in close temporal association, a new erection wasconsidered to occur when pressure dropped below FB+30mmHg for more than 15 seconds or when it droppedbelow FB+15 mmHg for more than 5 seconds. For statisticalpurposes, CSP pressure peaks greater than FB+80mmHg wereevaluated. Vascular latencywasmeasured as the time from thestart of the stimulus to the start of erection. Peak frequencieswere obtainedby dividing thenumber of peaks by the time fromthe first peak to the last peak. Data were analyzed using Spike 2data software (Cambridge Electronic Design, UK).

5.2.2. Data analysisThe Mann–Whitney U test with Bonferroni correction wasperformed to compare the medians of response values among

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response types (Table 1) or among stimulation sites. The χ2 testwas performed to compare the probability of each type ofresponse between anesthetized and unanesthetized groups.Statistica softwarewasused for theseanalyses.Differenceswereconsidered significant at p<0.05. Because the maximum valuemeasured by the transducer was 400 mmHg, the CSP peakssometimes exceeded thisvalue.ACSPpressure>400 mmHgwascounted as 400mmHg for purposes of statistical analysis.

Acknowledgments

This study was supported by Grant-in-Aid for ScientificResearch from Japan Society for the Promotion of Science toY. Koyama. The authors thank Ms. Nobuko Anzai for hertechnical assistance.

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