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Role of the lateral preoptic area and the bed nucleus of stria terminalis in the regulation of penile erection
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Research Report
Role of the lateral preoptic area and the bed nucleus of stria terminalis in the regulation of penile erection Hiroshi Iwasaki a,b , Eiichi Jodo a , Akihiro Kawauchi c , Tsuneharu Miki c , Yukihiko Kayama a , Yoshimasa Koyama d,⁎ a
Department of Neurophysiology, Fukushima Medical University, 1 Hikari-ga-oka, Fukushima 960-1295, Japan Department of Urology, Maizuru Kyosai Hospital, 1035, Hama, Maizuru, Kyoto 625-0037, Japan c Department of Urology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan d Department of Science and Technology, Fukushima University, 1-Kanaya-gawa, Fukushima 960-1296, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
To elucidate the role of the preoptic area (POA) in the regulation of penile erection, we
Accepted 3 August 2010
examined the effects of electrical stimulation in and around the POA on penile erection in rats,
Available online 10 August 2010
which was assessed by changes in pressure in the corpus spongiosum of the penis (CSP) and electromyography (EMG) of the bulbospongiosus (BS) muscle. In unanesthetized and
Keywords:
anesthetized rats, four types of responses were induced by stimulation in and around the
Penile erection
POA; (1) normal type responses, which were similar to spontaneously occurring erections,
Medial preoptic area
characterized by slow increase in CSP pressure and sharp peaks concurrent with BS muscle
Lateral preoptic area
bursting; (2) muscular type responses, which included sharp CSP pressure peaks (muscular
Bed nucleus of the stria terminalis
component) with almost no vascular component; (3) mixed type responses, which included a
Paraventricular nucleus
sequence of high-frequency CSP peaks followed by low-frequency CSP peaks; and (4) micturition type responses, which had higher-frequency and lower-amplitude CSP peaks than other responses which were identical to those of normal micturition. In unanesthetized condition, erections were evoked by stimulation of the lateral preoptic area (LPOA), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BST), paraventricular nucleus (PVN), reuniens thalamic nucleus (Re) and lateral septum (LS). Lower-intensity stimulation evoked erections 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 play different roles in mediating penile erection. © 2010 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Fax: + 81 24 548 8440. E-mail address: [email protected] (Y. Koyama). Abbreviations: BS, bulbospongiosus; BST, bed nucleus of the stria terminalis; CSP, corpus spongiosum of the penis; EEG, electroencephalogram; EMG, electromyogram; FB, flaccid baseline; LDT, laterodorsal tegmental nucleus; LPOA, lateral preoptic area; LS, lateral septum; MPOA, medial preoptic area; NCE, non-contact erection; PVN, paraventricular nucleus; Re, reuniens thalamic nucleus; REM, rapid eye movement 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.08.006
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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 in sexual function (Coolen, 2005). Electrical stimulation of the medial 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 MPOA caused severe deficits in copulation (Ginton and Merari, 1977; Hansen et al., 1982). Single neuronal activity in the MPOA exhibited a close correlation with male sexual behavior in rats and monkeys (Oomura et al., 1983; Shimura et al., 1994). Activation of the paraventricular nucleus (PVN) by NMDA injection caused penile erection (Melis et al., 1997). Lesions of the bed nucleus of the stria terminalis (BST) induced deficiency in some aspects of sexual behavior (Valcourt and Sachs, 1979; Liu et al., 1997a). Schmidt et al. (2000) have reported that ibotenic acid lesions of the lateral preoptic area (LPOA) disrupted penile erection during rapid eye movement (REM) sleep, while leaving waking-state erections intact. These reports suggest that there are two regulatory systems for penile erection located in the POA; the MPOA system, which is involved in sexual behaviorrelated erection, and the LPOA system, which is crucial for sleeprelated erection. To test this hypothesis, we examined the effects of electrical stimulation in and around the POA on penile erection in rats. For this experiment, we used fine carbon fiber electrodes which are movable and able to systematically stimulate numerous points in one animal, and measured erectile tissue pressure using a telemetric monitoring technique (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 area yielded four different types of responses; normal, muscular type, mixed type and micturition type. Fig. 1 shows an
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example of a normal response evoked by delivery of a stimulus of 100 μA to the lateral preoptic area (LPOA). This stimulus induced, with a latency of 20 seconds, a slow increase in CSP pressure reaching 30 mmHg and sharp CSP pressure peaks riding on the slow CSP pressure increase. The BS muscle exhibited bursting discharges in accordance with the CSP pressure peaks. This pattern of erection was similar to that of spontaneous erections. Fig. 2 shows the profile of the responses obtained at different depths of a track passing through 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 0.4mm deeper (Trace B). Although small BS muscle activity was evoked at depths of 7.8 mm and 8.2 mm, no erections were evoked (Traces C and D, respectively). At 8.6 mm from the surface, a 50-μA stimulus evoked erection (Trace F). However, with a stimulus of 50 μA, no response was observed at points 0.2 mm away (dorsal and ventral) from the effective point (Traces E and G). These findings indicate that the effects of a 50-μA stimulus do not spread further than 0.2 mm, while those of a 100-μA stimulus are limited to 0.4 mm from the stimulation point. As shown in Fig. 3, the muscular type response, similar to normal responses, exhibited a series of sharp CSP pressure peaks with a characteristically small vascular component (less than 30 mmHg). The sharp CSP pressure peaks occurred simultaneously with BS muscle activity (they are not shown in Fig. 3, since in this preparation the recording wires for BS muscle were removed). The sharp CSP pressure peaks appeared more regularly than those in normal responses. The coefficient of variation of the peak interval in muscular type responses, including those in both unanesthetized and anesthetized rats, was significantly smaller than that for normal responses (p < 0.01 by Mann–Whitney U test). Fig. 4 shows an example of a mixed type response in which, in the vascular component, two types of CSP pressure peaks occur sequentially; initial high-frequency peaks (phase A) followed by low-frequency peaks (phase B). As Table 1 indicates, the peak frequencies of phase A were significantly higher than those in normal responses (p < 0.001) and muscular type responses (p < 0.001), while the peak frequencies of
Fig. 1 – Normal response after stimulation (100 μA) of the lateral preoptic area, which caused a CSP pressure pattern similar to that of a spontaneous erection and BS muscle activity synchronous with CSP pressure peaks. BS, bulbospongiosus; CSP, corpus spongiosum of the penis; EMG, electromyogram; EEG, electroencephalogram.
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than those of normal, muscular type and mixed type responses (phase B) (p < 0.05). Of 67 responses obtained in unanesthetized 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 responses were evoked on stimulation in unanesthetized animals. Of 364 sites in and around the preoptic and hypothalamic areas, responses were evoked from 39 sites including the lateral preoptic area (LPOA), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BST) and paraventricular nucleus (PVN). In addition, stimulation in surrounding areas including the reuniens thalamic nucleus (Re) and the lateral septum (LS) was also effective. Stimulation in the thalamus, anterior hypothalamus and lateral hypothalamus was not effective. The ratios of effective sites to stimulation sites in the LPOA, 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 stimulus intensity of less than 50 μA, erections were more effectively evoked from the BST at the level of Br 0.26 to Br 0.40, posterior half of the LPOA (Br 0.92 to Br 1.30) and the PVN (Br 1.30). On the other hand, in the case of responses obtained from the MPOA, stimulus intensities above 100 μA (in most cases, 200 μA or 300 μA) were required.
Fig. 2 – Depth profiles of effects of electrical stimulation at depths of 7.0 mm (A) to 8.8 mm (G) from the brain surface. Circles, effective points; crosses, ineffective points; stim, stimulation.
2.2.
phase B were within the range of normal responses and muscular type responses. In addition, the peak-to-peak duration of phase A was significantly shorter than those of normal type (p < 0.05) and muscular type (p < 0.05) responses. The response shown in Fig. 5 consisted of regular, highfrequency, low-amplitude CSP peaks. The peak frequency was from 9.7 to 10.3 Hz, and within the range observed in our previous study (Yamao et al., 2001) or close to the discharge frequency of the sphincter muscle discharge during naturally occurring micturition (Nout et al., 2007). This response was therefore termed micturition type response. The peak frequency of the micturition type response was the highest of all responses (p < 0.001), and the high-frequency peaks were synchronous with BS muscle bursts. In micturition type responses, the maximum peak amplitudes were smaller
Under urethane anesthesia, four types of responses – normal, muscular type, mixed type and micturition type – were observed (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 unanesthetized condition, the proportion of muscular type responses was significantly higher (p < 0.02 by χ2 test.) In anesthetized condition, effective sites were restricted to the LPOA, MPOA and BST (Fig. 6B, Table 2). The ratios of effective sites to stimulation sites in the LPOA, MPOA and BST were 8/170 (4.7%), 13/200 (6.5%) and 4/78 (5.0%), respectively. The ratios were lower than those in unanesthetized condition (13/131 (10%), 7/55 (12.7%) and 8/94 (8.5%), respectively). In total, responsiveness under urethane anesthesia (25/561, 4.5%) was significantly lower than in unanesthetized condition (39/364, 10.7%) (Table 2). Stronger stimuli (more than 100 μA) were required to elicit erection than
Responses in anesthetized condition
Fig. 3 – Muscular type response after stimulation (300 μA) of the paraventricular nucleus, with a CSP pressure pattern with almost no vascular component but accompanied by steep periodic CSP peaks. Since the BS EMG could not be recorded, the trace was deleted from the figure.
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Fig. 4 – Mixed type response after stimulation (50 μA) of the lateral preoptic area, which is composed of a CSP pressure pattern with high-frequency CSP peaks (Phase A) followed by low-frequency CSP peaks (Phase B).
in the unanesthetized condition. Compared with unanesthetized condition, latencies were longer (p < 0.01), the frequency and 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 response (0.02–0.52, 0.25 Hz) were significantly lower than those of muscular type responses (0.31–1.33, 0.62 Hz) (p < 0.01) (Table 1).
3.
Discussion
In rats in unanesthetized and anesthetized conditions, four types of responses – normal, muscular type, mixed type, and micturition type – were induced by stimulation in and around the preoptic area. Normal responses were most similar to spontaneously occurring erections. Normal responses were composed of a slow increase in CSP pressure and sharp peaks concurrent with BS muscle bursting. The amplitude, frequency and duration of these responses were within the ranges observed in our previous studies for spontaneous erections or normal erections (Gulia et al., 2008; Salas et al., 2007). The evoked response was thus quite similar to physiological erection. Induction of muscular type responses, with sharp CSP pressure peaks (muscular component) with almost no vascular component, suggests that in the preoptic area the
neural system regulating the muscular component is separate from that regulating the vascular component. Electrical stimulation often induced slow CSP pressure increase (vascular component) without, or with only a single, sharp CSP peak (unpublished observation), suggesting that the neural system regulating the vascular component was selectively activated. Induction of mixed type responses, which included a sequence of high-frequency CSP peaks followed by low-frequency CSP peaks, suggested that two systems were activated, one, generating high-frequency BS muscle activity and the other generating BS muscle activity similar to that observed in normal responses or spontaneous erections. Micturition type responses had higher-frequency and lower-amplitude CSP peaks than other responses. The peak frequency (5.16– 10.29 Hz) covered the range of the frequency of the external sphincter muscle discharges during micturition (Kruse et al., 1990; Yamao et al., 2001). In some cases, we observed voiding when the high-frequency CSP fluctuation occurred (unpublished observation). Micturition type responses thus appear to reflect normal physiological micturition. The micturition center in the brainstem (Barrington's nucleus) receives projection from the preoptic area (Rizvi et al., 1994). The stimuli that induced micturition type responses were thus transmitted to Barrington's nucleus through the preopticBarrington's nucleus pathway.
Table 1 – Parameters of four types of stimulus-evoked responses.
*p < 0.05; **p < 0.01; ***p < 0.001 by Mann–Whitney U test with correction.
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Fig. 5 – Micturition type response, evoked by stimulation (50 μA) of the lateral septum and composed of high-frequency CSP pressure peaks without a vascular component.
Fig. 6 – Coronal sections showing the location of effective sites from which each type of response was evoked. (A) Unanesthetized condition; (B) anesthetized condition. Red circles, normal responses; yellow circles, muscular type responses; blue circles, mixed type responses; green circles, micturition type responses; circles with multiple colors, sites from which two or three types of responses were evoked; large circles, responses evoked by less than 50 μA stimulation; medium-sized diamonds, responses evoked by less than 100 μA stimulation; small circles, responses evoked by less than 300 μA stimulation. AC, anterior commissure; BST, bed nucleus of 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 in each anatomical area. Stimulation sites LPOA MPOA BST PVN RE LS TH AH LH Total
Positive sites/total sites Unanesthetized 13/131 7/55 8/94 4/10 3/11 4/21 0/33 0/4 0/5 39/364
(0.099) (0.127) (0.085) (0.4) (0.272) (0.19) (0) (0) (0) (0.107)
Anesthetized 8/170 (0.047) 13/200 (0.065) 4/78 (0.05) 0/13 (0) 0/9 (0) 0/10 (0) 0/60 (0) 0/17 (0) 0/4 (0) 25/561 (0.045)
In unanesthetized condition, the proportions of normal, muscular type, mixed type and micturition type responses were 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 while that 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 the mesopontine tegmentum is more closely related to micturition than the preoptic area. The values obtained in the present study were similar to those obtained in the septum stimulation study, in which the frequency of normal responses was higher (89.0%; 65/73) than those of other responses (mixed type [6.8%; 5/73] and micturition type [4.1%; 3/73]) (Gulia et al., 2008). Under anesthetized condition, compared with unanesthetized condition, latencies were longer (p < 0.01), the frequency and 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 a facilitatory role in penile erection (Aioun and Rampin, 2006; Song and Rajasekaran, 2004; Succu et al., 2006), and that urethane suppresses the function of NMDA receptor (Hara and Harris, 2002). Suppression of NMDA receptors by urethane would thus have caused longer latencies and lower peak frequencies. It is also possible that, in normal spontaneous erections, inhibitory mechanisms are at work to terminate erection. Urethane anesthesia would have suppressed such termination signals due to such mechanisms, resulting in longer peak-to-peak duration. Under anesthetized condition, muscular type responses were observed frequently, indicating that urethane anesthesia more strongly suppressed the system activating the vascular component. Under unanesthetized condition, erections were evoked from the LPOA, MPOA, BST, PVN, Re and LS, while under anesthetized condition, stronger stimuli were required, and effective sites were restricted to the LPOA, MPOA and BST. Under urethane anesthesia, erection was thus evoked only from the crucial areas for regulating erection. Schmidt et al. reported that ibotenic acid lesions of LPOA disrupted penile erection during REM sleep, while leaving waking-condition erection intact, and suggested that the LPOA plays a crucial role in penile erection during REM sleep (Schmidt et al., 2000). Our finding that penile erections were evoked by
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stimulation of the LPOA further confirms that the neural substrate for penile erection is located in the LPOA. Schmidt et al. (2001) have also reported that injections of carbachol in the LPOA induce penile erections. We found that stimulation of the laterodorsal tegmental nucleus (LDT, one of the brainstem cholinergic nuclei) induced erection (Salas et al., 2007), and suggested that the LDT is a cholinergic source for induction of erection in the LPOA. The MPOA is known to play crucial roles in the regulation of reproductive 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 in penile erection. Schmidt et al. (2000) found that MPOA lesions had almost no effect on waking-state or REM sleep-related erection. Liu et al., (1997a) have reported that male rats with MPOA lesions exhibit severe deficits in copulation but little or no decrement in non-contact erection (NCE), while BST lesions cause impairment of NCE with only slight deficits in copulation, suggesting that the BST plays an important role in mediating NCE. In the present study, although erections were evoked from the MPOA, the stimulus intensities required for them were higher than those for elicitation of erection from the LPOA or BST. The MPOA thus appears to play a role in penile erection different from those of the LPOA and BST. Considering the spread of stimulus current (Fig. 2), it is possible that the stimulation of higher intensity applied to the MPOA invaded the LPOA or BST. The neurons in the BST send projections to the LPOA and MPOA (Dong and Swanson, 2006). Liu et al. (1997a) have suggested that an extra-MPOA pathway mediates NCE. The system extending from BST to LPOA might correspond to this pathway. 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), while lesions of PVN disrupts NCE (Liu et al., 1997b; Melis et al., 2001). The PVN projects directly to the intermediolateral region of the spinal cord (Ranson et al., 1998). In the present study, erection was evoked by PVN stimulation with lower intensities than the MPOA (Fig. 6A) and higher probability than any other brain areas (Table 2). These facts support the idea that PVN neurons receiving dopaminergic or glutamatergic inputs and projecting directly to the spinal cord act as the final output of the erection regulating system in the hypothalamus.
4.
Conclusion
In unanesthetized rats, penile erection was evoked by the electrical stimulation of the LPOA, MPOA, BST, PVN RE and LS. Among them, lower-intensity stimulus was effective in the LPOA, BST, PVN and RE, but not in the MPOA. In anesthetized condition, the effective areas were restricted to the LPOA, MPOA and BST. The present results suggest the difference of the lateral and medial subdivisions of the POA, emphasizing the role of the lateral subdivisions (LPOA and BST), in the regulation 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 a 12:12-h light/dark cycle at an ambient temperature of 22.0 ± 0.1 °C with food and water available ad libitum. Of them, 13 were used under urethane anesthesia, while 7 were used unanesthetized in head-restrained condition. All procedures were carried out under the control of the Animal Research Committee in accordance with the Guidelines for Animal Experiments of Fukushima Medical University and the Japanese Animal Protection and Management Law (No. 105). All efforts were made to minimize the number of animals used and their suffering.
5.1.1.
Surgical procedure
For acute experiments, animals were anesthetized with urethane (1.2 g/ kg, i.p.). For surgery for chronic experiments, sodium pentobarbital (50 mg/kg) was intraperitoneally injected and additional doses were given to maintain the level of anesthesia. As previously described, the pressure of the corpus spongiosum of the penis (CSP) was measured telemetrically (Gulia et al., 2008; Nout et al., 2007; Schmidt et al., 1994; Salas et al., 2007). In brief, the distal portion of the bulb of the corpus spongiosum of the penis (CSP) was gently exposed and the catheter tip of the telemetric transducer (TA11PA-C40, Data Sciences International, St. Paul MN) was inserted into the bulb through a slit made by a needle. The body of the transducer was fixed subcutaneously to the abdominal muscle. The catheter was secured using a biological glue (Vetbond, 3 M Animal Care Products) at the point of entrance of the catheter and was sutured to the fascia overlying the shaft of the penis. To record the electromyogram (EMG) of the bulbospongiosus (BS) muscle, a pair of stainless wires were inserted into the BS muscle and passed subcutaneously to an incision of the skin over the skull which had been made in advance of the implantation. To assess vigilance state, stainless steel screws (tip diameter= 1.4 mm) for recording of the cortical electroencephalogram (EEG) were implanted in the skull overlying the frontal and parietal cortices and, in unanesthetized, head-restrained rats, wire electrodes were implanted in the neck muscle to record the electromyogram (EMG). For painless fixation of the unanesthetized rats to a stereotaxic frame during experiments, a U-shaped plastic plate (width 20 mm, length 30 mm, thickness 5 mm) was attached to the 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 rats were given a 1-week period for recovery.
5.2.
Experimental procedures
One day before the experiment, rats were attached to the stereotaxic frame under ketamine anesthesia (50 mg/kg) using the U-shaped plate, and a small hole was made through the occipital bone using a dental drill to expose the surface of the cerebral cortex above the preoptic area. The rats were deprived of sleep for 12 h in a slowly rotating wheel (diameter 37 mm, width 10 cm, 1.2 rev/min) with free access to food and water. On
the day of the experiment, rats were returned to their cage to rest for 2–3 h. They were then fixed to the stereotaxic frame using the U-shaped plate. The stimulation electrode consisted of a glass pipette which had a carbon fiber (diameter, <10 μm; resistance, 4–6 MΩ) protruding from the tip about 20–30 μm and a cavity filled with Woods metal (Takakusaki et al., 1989). To determine sites most effective for induction of erection, stimulation was applied, usually at 0.2 mm intervals, by moving the electrode with an oil microdrive manipulator. Stimuli were given 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 leg movement. Therefore, in most cases the stimuli were limited to less than 100 μA. The most effective site along the track was marked by passing 20 μA positive current (DC) for 30 s. After completion of the experiment, the rats were deeply anesthetized with pentobarbital and perfused transcardially with 300 ml of physiological saline followed by 300 ml of 10% formalin. The brain was then removed, postfixed in the same fixative, soaked in 30% sucrose and sectioned in the coronal plane at a thickness of 50 μm. The sections were stained with neutral red. Stimulation sites were identified under light microscopy using standard nomenclature according to the rat brain atlas of Paxinos and Watson (1997).
5.2.1.
Definition of responses
As shown in Fig. 1, erection is composed of a slow CSP pressure increase and sharp CSP pressure peaks concurrent with bursting BS muscle activity. The slow CSP pressure increase reflects the flow of blood into the CSP caused by the distention of the blood vessels supplying the CSP, and is termed the vascular component, while the sharp peaks reflect each of the erectile events of the penis (flip), which are caused by bursting discharges of the BS muscles (muscular component). In the rat, the vascular component is associated with an increase in the baseline erectile tissue pressure from approximately 10–15 mmHg in the flaccid state (Flaccid Baseline, FB) to a tumescence pressure up to approximately 50–70 mmHg. The muscular component is easily identified by the sharp, suprasystolic CSP pressure peaks occurring on top of the tumescence pressure. Normal erection was defined as a tumescence pressure at least 30 mmHg higher than the FB. The start of erection was defined as the time the CSP pressure reached FB+ 30 mmHg, while the end of erection was defined as the time when CSP pressure fell below FB+ 30 mmHg for more than 15 s. To distinguish between two erections occurring in close temporal association, a new erection was considered to occur when pressure dropped below FB + 30 mmHg for more than 15 seconds or when it dropped below FB+ 15 mmHg for more than 5 seconds. For statistical purposes, CSP pressure peaks greater than FB + 80 mmHg were evaluated. Vascular latency was measured as the time from the start of the stimulus to the start of erection. Peak frequencies were obtained by dividing the number of peaks by the time from the first peak to the last peak. Data were analyzed using Spike 2 data software (Cambridge Electronic Design, UK).
5.2.2.
Data analysis
The Mann–Whitney U test with Bonferroni correction was performed to compare the medians of response values among
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response types (Table 1) or among stimulation sites. The χ2 test was performed to compare the probability of each type of response between anesthetized and unanesthetized groups. Statistica software was used for these analyses. Differences were considered significant at p < 0.05. Because the maximum value measured by the transducer was 400 mmHg, the CSP peaks sometimes exceeded this value. A CSP pressure > 400 mmHg was counted as 400 mmHg for purposes of statistical analysis.
Acknowledgments This study was supported by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science to Y. Koyama. The authors thank Ms. Nobuko Anzai for her technical assistance.
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oxytocin-induced yawning and penile erection. Neurosci Res. 54, 269–275. Kruse, M.N., Noto, H., Roppolo, J.R., de Groat, W.C., 1990. Pontine control of the urinary bladder and external urethral sphincter in the rat. Brain Res. 532, 182–190. Liu, Y.C., Salamone, J.D., Sachs, B.D., 1997a. Lesions in medial preoptic area and bed nucleus of stria terminalis: differential effects on copulatory behavior and noncontact erection in male rats. J Neurosci. 17, 5245–5253. Liu, Y.C., Salamone, J.D., Sachs, B.D., 1997b. Impaired sexual response after lesions of the paraventricular nucleus of the hypothalamus in male rats. Behav Neurosci. 111, 1361–1367. Malsbury, C.W., 1971. Facilitation of male rat copulatory behavior by electrical stimulation of the medial preoptic area. Physiol Behav. 7, 797–805. Melis, M.R., Argiolas, A., Gessa, G.L., 1987. Apomorphine-induced penile erection and yawning: site of action in brain. Brain Res. 415, 98–104. Melis, M.R., Succu, S., Iannucci, U., Argiolas, A., 1997. N-methyl-D-aspartic acid-induced penile erection and yawning: role of hypothalamic paraventricular nitric oxide Eur J Pharmacol. 328, 115–123. Melis, M.R., Succu, S., Mascia, M.S., Argiolas, A., 2001. The activation of gamma aminobutyric acid(A) receptors in the paraventricular nucleus of the hypothalamus reduces non-contact penile erections in male rats. Neurosci Lett. 314, 123–126. Merari, A., Ginton, A., 1975. Characteristics of exaggerated sexual behavior induced by electrical stimulation of the medial preoptic area in male rats. Brain Res. 86, 97–108. Nout, Y.S., Bresnahan, J.C., Culp, E., Tovar, C.A., Beattie, M.S., Schmidt, M.H., 2007. Novel technique for monitoring micturition and sexual function in male rats using telemetry. Am J Physiol Regul Integr Comp Physiol. 292, R1359–R1367. Oomura, Y., Yoshimatsu, H., Aou, S., 1983. Medial preoptic and hypothalamic neuronal activity during sexual behavior of the male monkey. Brain Res. 266, 340–343. Paxinos, G., Watson, C., 1997. The rat brain in stereotaxic coordinates. Academic Press, San Diego, CA. Ranson, R.N., Motawei, K., Pyner, S., Coote, J.H., 1998. The paraventricular nucleus of the hypothalamus sends efferents to the spinal cord of the rat that closely appose sympathetic preganglionic neurones projecting to the stellate ganglion. Exp Brain Res. 120, 164–172. Rizvi, T.A., Ennis, M., Aston-Jones, G., Jiang, M., Liu, W.L., Behbehani, M.M., Shipley, M.T., 1994. Preoptic projections to Barrington's nucleus and the pericoerulear region: architecture and terminal organization. J Comp Neurol. 347, 1–24. Salas, J.C., Iwasaki, H., Jodo, E., Schmidt, M.H., Kawauchi, A., Miki, T., Kayama, Y., Otsuki, M., Koyama, Y., 2007. Penile erection and micturition events triggered by electrical stimulation of the mesopontine tegmental area. Am J Physiol Regul Integr Comp Physiol. 294, R102–R111. Sato, Y., Christ, G.J., 2000. Differential ICP responses elicited by electrical stimulation of medial preoptic area. Am J Physiol Heart Circ Physiol. 278, H964–H970. Schmidt MH, Gervasoni D, Luppi P, Fort P., 2001. Carbachol administration into the lateral preoptic area induces erections and wakefulness. In: Abstract viewer/Itinerary Planner. the Society for Neuroscience 31st Annual Meeting. San Diego, CA: Progr. No 522.19. Schmidt, M.H., Valatx, J.L., Sakai, K., Fort, P., Jouvet, M., 2000. Role of the lateral preoptic area in sleep-related erectile mechanisms and sleep generation in the rat. J Neurosci. 20, 6640–6647. Schmidt, M.H., Valatx, J.L., Schmidt, H.S., Wauquier, A., Jouvet, M., 1994. Experimental evidence of penile erections during paradoxical sleep in the rat. Neuroreport. 5, 561–564. Shimura, T., Yamamoto, T., Shimokochi, M., 1994. The medial preoptic area is involved in both sexual arousal
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and performance in male rats: re-evaluation of neuron activity in freely moving animals. Brain Res. 640, 215–222. Song, Y., Rajasekaran, M., 2004. Effect of excitatory amino acid receptor agonists on penile erection after administration into the CA3 hippocampal region in the rat. Urology. 64, 1250–1254. Succu, S., Mascia, M.S., Sanna, F., Melis, T., Argiolas, A., Melis, M.R., 2006. The cannabinoid CB1 receptor antagonist SR 141716A induces penile erection by increasing extra-cellular glutamic acid in the paraventricular nucleus of male rats. Behav Brain Res. 169, 274–281.
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Research Report
Role of the lateral preoptic area and the bed nucleus of stria terminalis in the regulation of penile erection Hiroshi Iwasaki a,b , Eiichi Jodo a , Akihiro Kawauchi c , Tsuneharu Miki c , Yukihiko Kayama a , Yoshimasa Koyama d,⁎ a
Department of Neurophysiology, Fukushima Medical University, 1 Hikari-ga-oka, Fukushima 960-1295, Japan Department of Urology, Maizuru Kyosai Hospital, 1035, Hama, Maizuru, Kyoto 625-0037, Japan c Department of Urology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan d Department of Science and Technology, Fukushima University, 1-Kanaya-gawa, Fukushima 960-1296, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
To elucidate the role of the preoptic area (POA) in the regulation of penile erection, we
Accepted 3 August 2010
examined the effects of electrical stimulation in and around the POA on penile erection in rats,
Available online 10 August 2010
which was assessed by changes in pressure in the corpus spongiosum of the penis (CSP) and electromyography (EMG) of the bulbospongiosus (BS) muscle. In unanesthetized and
Keywords:
anesthetized rats, four types of responses were induced by stimulation in and around the
Penile erection
POA; (1) normal type responses, which were similar to spontaneously occurring erections,
Medial preoptic area
characterized by slow increase in CSP pressure and sharp peaks concurrent with BS muscle
Lateral preoptic area
bursting; (2) muscular type responses, which included sharp CSP pressure peaks (muscular
Bed nucleus of the stria terminalis
component) with almost no vascular component; (3) mixed type responses, which included a
Paraventricular nucleus
sequence of high-frequency CSP peaks followed by low-frequency CSP peaks; and (4) micturition type responses, which had higher-frequency and lower-amplitude CSP peaks than other responses which were identical to those of normal micturition. In unanesthetized condition, erections were evoked by stimulation of the lateral preoptic area (LPOA), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BST), paraventricular nucleus (PVN), reuniens thalamic nucleus (Re) and lateral septum (LS). Lower-intensity stimulation evoked erections 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 play different roles in mediating penile erection. © 2010 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Fax: + 81 24 548 8440. E-mail address: [email protected] (Y. Koyama). Abbreviations: BS, bulbospongiosus; BST, bed nucleus of the stria terminalis; CSP, corpus spongiosum of the penis; EEG, electroencephalogram; EMG, electromyogram; FB, flaccid baseline; LDT, laterodorsal tegmental nucleus; LPOA, lateral preoptic area; LS, lateral septum; MPOA, medial preoptic area; NCE, non-contact erection; PVN, paraventricular nucleus; Re, reuniens thalamic nucleus; REM, rapid eye movement 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.08.006
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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 in sexual function (Coolen, 2005). Electrical stimulation of the medial 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 MPOA caused severe deficits in copulation (Ginton and Merari, 1977; Hansen et al., 1982). Single neuronal activity in the MPOA exhibited a close correlation with male sexual behavior in rats and monkeys (Oomura et al., 1983; Shimura et al., 1994). Activation of the paraventricular nucleus (PVN) by NMDA injection caused penile erection (Melis et al., 1997). Lesions of the bed nucleus of the stria terminalis (BST) induced deficiency in some aspects of sexual behavior (Valcourt and Sachs, 1979; Liu et al., 1997a). Schmidt et al. (2000) have reported that ibotenic acid lesions of the lateral preoptic area (LPOA) disrupted penile erection during rapid eye movement (REM) sleep, while leaving waking-state erections intact. These reports suggest that there are two regulatory systems for penile erection located in the POA; the MPOA system, which is involved in sexual behaviorrelated erection, and the LPOA system, which is crucial for sleeprelated erection. To test this hypothesis, we examined the effects of electrical stimulation in and around the POA on penile erection in rats. For this experiment, we used fine carbon fiber electrodes which are movable and able to systematically stimulate numerous points in one animal, and measured erectile tissue pressure using a telemetric monitoring technique (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 area yielded four different types of responses; normal, muscular type, mixed type and micturition type. Fig. 1 shows an
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example of a normal response evoked by delivery of a stimulus of 100 μA to the lateral preoptic area (LPOA). This stimulus induced, with a latency of 20 seconds, a slow increase in CSP pressure reaching 30 mmHg and sharp CSP pressure peaks riding on the slow CSP pressure increase. The BS muscle exhibited bursting discharges in accordance with the CSP pressure peaks. This pattern of erection was similar to that of spontaneous erections. Fig. 2 shows the profile of the responses obtained at different depths of a track passing through 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 0.4mm deeper (Trace B). Although small BS muscle activity was evoked at depths of 7.8 mm and 8.2 mm, no erections were evoked (Traces C and D, respectively). At 8.6 mm from the surface, a 50-μA stimulus evoked erection (Trace F). However, with a stimulus of 50 μA, no response was observed at points 0.2 mm away (dorsal and ventral) from the effective point (Traces E and G). These findings indicate that the effects of a 50-μA stimulus do not spread further than 0.2 mm, while those of a 100-μA stimulus are limited to 0.4 mm from the stimulation point. As shown in Fig. 3, the muscular type response, similar to normal responses, exhibited a series of sharp CSP pressure peaks with a characteristically small vascular component (less than 30 mmHg). The sharp CSP pressure peaks occurred simultaneously with BS muscle activity (they are not shown in Fig. 3, since in this preparation the recording wires for BS muscle were removed). The sharp CSP pressure peaks appeared more regularly than those in normal responses. The coefficient of variation of the peak interval in muscular type responses, including those in both unanesthetized and anesthetized rats, was significantly smaller than that for normal responses (p < 0.01 by Mann–Whitney U test). Fig. 4 shows an example of a mixed type response in which, in the vascular component, two types of CSP pressure peaks occur sequentially; initial high-frequency peaks (phase A) followed by low-frequency peaks (phase B). As Table 1 indicates, the peak frequencies of phase A were significantly higher than those in normal responses (p < 0.001) and muscular type responses (p < 0.001), while the peak frequencies of
Fig. 1 – Normal response after stimulation (100 μA) of the lateral preoptic area, which caused a CSP pressure pattern similar to that of a spontaneous erection and BS muscle activity synchronous with CSP pressure peaks. BS, bulbospongiosus; CSP, corpus spongiosum of the penis; EMG, electromyogram; EEG, electroencephalogram.
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than those of normal, muscular type and mixed type responses (phase B) (p < 0.05). Of 67 responses obtained in unanesthetized 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 responses were evoked on stimulation in unanesthetized animals. Of 364 sites in and around the preoptic and hypothalamic areas, responses were evoked from 39 sites including the lateral preoptic area (LPOA), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BST) and paraventricular nucleus (PVN). In addition, stimulation in surrounding areas including the reuniens thalamic nucleus (Re) and the lateral septum (LS) was also effective. Stimulation in the thalamus, anterior hypothalamus and lateral hypothalamus was not effective. The ratios of effective sites to stimulation sites in the LPOA, 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 stimulus intensity of less than 50 μA, erections were more effectively evoked from the BST at the level of Br 0.26 to Br 0.40, posterior half of the LPOA (Br 0.92 to Br 1.30) and the PVN (Br 1.30). On the other hand, in the case of responses obtained from the MPOA, stimulus intensities above 100 μA (in most cases, 200 μA or 300 μA) were required.
Fig. 2 – Depth profiles of effects of electrical stimulation at depths of 7.0 mm (A) to 8.8 mm (G) from the brain surface. Circles, effective points; crosses, ineffective points; stim, stimulation.
2.2.
phase B were within the range of normal responses and muscular type responses. In addition, the peak-to-peak duration of phase A was significantly shorter than those of normal type (p < 0.05) and muscular type (p < 0.05) responses. The response shown in Fig. 5 consisted of regular, highfrequency, low-amplitude CSP peaks. The peak frequency was from 9.7 to 10.3 Hz, and within the range observed in our previous study (Yamao et al., 2001) or close to the discharge frequency of the sphincter muscle discharge during naturally occurring micturition (Nout et al., 2007). This response was therefore termed micturition type response. The peak frequency of the micturition type response was the highest of all responses (p < 0.001), and the high-frequency peaks were synchronous with BS muscle bursts. In micturition type responses, the maximum peak amplitudes were smaller
Under urethane anesthesia, four types of responses – normal, muscular type, mixed type and micturition type – were observed (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 unanesthetized condition, the proportion of muscular type responses was significantly higher (p < 0.02 by χ2 test.) In anesthetized condition, effective sites were restricted to the LPOA, MPOA and BST (Fig. 6B, Table 2). The ratios of effective sites to stimulation sites in the LPOA, MPOA and BST were 8/170 (4.7%), 13/200 (6.5%) and 4/78 (5.0%), respectively. The ratios were lower than those in unanesthetized condition (13/131 (10%), 7/55 (12.7%) and 8/94 (8.5%), respectively). In total, responsiveness under urethane anesthesia (25/561, 4.5%) was significantly lower than in unanesthetized condition (39/364, 10.7%) (Table 2). Stronger stimuli (more than 100 μA) were required to elicit erection than
Responses in anesthetized condition
Fig. 3 – Muscular type response after stimulation (300 μA) of the paraventricular nucleus, with a CSP pressure pattern with almost no vascular component but accompanied by steep periodic CSP peaks. Since the BS EMG could not be recorded, the trace was deleted from the figure.
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Fig. 4 – Mixed type response after stimulation (50 μA) of the lateral preoptic area, which is composed of a CSP pressure pattern with high-frequency CSP peaks (Phase A) followed by low-frequency CSP peaks (Phase B).
in the unanesthetized condition. Compared with unanesthetized condition, latencies were longer (p < 0.01), the frequency and 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 response (0.02–0.52, 0.25 Hz) were significantly lower than those of muscular type responses (0.31–1.33, 0.62 Hz) (p < 0.01) (Table 1).
3.
Discussion
In rats in unanesthetized and anesthetized conditions, four types of responses – normal, muscular type, mixed type, and micturition type – were induced by stimulation in and around the preoptic area. Normal responses were most similar to spontaneously occurring erections. Normal responses were composed of a slow increase in CSP pressure and sharp peaks concurrent with BS muscle bursting. The amplitude, frequency and duration of these responses were within the ranges observed in our previous studies for spontaneous erections or normal erections (Gulia et al., 2008; Salas et al., 2007). The evoked response was thus quite similar to physiological erection. Induction of muscular type responses, with sharp CSP pressure peaks (muscular component) with almost no vascular component, suggests that in the preoptic area the
neural system regulating the muscular component is separate from that regulating the vascular component. Electrical stimulation often induced slow CSP pressure increase (vascular component) without, or with only a single, sharp CSP peak (unpublished observation), suggesting that the neural system regulating the vascular component was selectively activated. Induction of mixed type responses, which included a sequence of high-frequency CSP peaks followed by low-frequency CSP peaks, suggested that two systems were activated, one, generating high-frequency BS muscle activity and the other generating BS muscle activity similar to that observed in normal responses or spontaneous erections. Micturition type responses had higher-frequency and lower-amplitude CSP peaks than other responses. The peak frequency (5.16– 10.29 Hz) covered the range of the frequency of the external sphincter muscle discharges during micturition (Kruse et al., 1990; Yamao et al., 2001). In some cases, we observed voiding when the high-frequency CSP fluctuation occurred (unpublished observation). Micturition type responses thus appear to reflect normal physiological micturition. The micturition center in the brainstem (Barrington's nucleus) receives projection from the preoptic area (Rizvi et al., 1994). The stimuli that induced micturition type responses were thus transmitted to Barrington's nucleus through the preopticBarrington's nucleus pathway.
Table 1 – Parameters of four types of stimulus-evoked responses.
*p < 0.05; **p < 0.01; ***p < 0.001 by Mann–Whitney U test with correction.
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Fig. 5 – Micturition type response, evoked by stimulation (50 μA) of the lateral septum and composed of high-frequency CSP pressure peaks without a vascular component.
Fig. 6 – Coronal sections showing the location of effective sites from which each type of response was evoked. (A) Unanesthetized condition; (B) anesthetized condition. Red circles, normal responses; yellow circles, muscular type responses; blue circles, mixed type responses; green circles, micturition type responses; circles with multiple colors, sites from which two or three types of responses were evoked; large circles, responses evoked by less than 50 μA stimulation; medium-sized diamonds, responses evoked by less than 100 μA stimulation; small circles, responses evoked by less than 300 μA stimulation. AC, anterior commissure; BST, bed nucleus of 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 in each anatomical area. Stimulation sites LPOA MPOA BST PVN RE LS TH AH LH Total
Positive sites/total sites Unanesthetized 13/131 7/55 8/94 4/10 3/11 4/21 0/33 0/4 0/5 39/364
(0.099) (0.127) (0.085) (0.4) (0.272) (0.19) (0) (0) (0) (0.107)
Anesthetized 8/170 (0.047) 13/200 (0.065) 4/78 (0.05) 0/13 (0) 0/9 (0) 0/10 (0) 0/60 (0) 0/17 (0) 0/4 (0) 25/561 (0.045)
In unanesthetized condition, the proportions of normal, muscular type, mixed type and micturition type responses were 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 while that 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 the mesopontine tegmentum is more closely related to micturition than the preoptic area. The values obtained in the present study were similar to those obtained in the septum stimulation study, in which the frequency of normal responses was higher (89.0%; 65/73) than those of other responses (mixed type [6.8%; 5/73] and micturition type [4.1%; 3/73]) (Gulia et al., 2008). Under anesthetized condition, compared with unanesthetized condition, latencies were longer (p < 0.01), the frequency and 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 a facilitatory role in penile erection (Aioun and Rampin, 2006; Song and Rajasekaran, 2004; Succu et al., 2006), and that urethane suppresses the function of NMDA receptor (Hara and Harris, 2002). Suppression of NMDA receptors by urethane would thus have caused longer latencies and lower peak frequencies. It is also possible that, in normal spontaneous erections, inhibitory mechanisms are at work to terminate erection. Urethane anesthesia would have suppressed such termination signals due to such mechanisms, resulting in longer peak-to-peak duration. Under anesthetized condition, muscular type responses were observed frequently, indicating that urethane anesthesia more strongly suppressed the system activating the vascular component. Under unanesthetized condition, erections were evoked from the LPOA, MPOA, BST, PVN, Re and LS, while under anesthetized condition, stronger stimuli were required, and effective sites were restricted to the LPOA, MPOA and BST. Under urethane anesthesia, erection was thus evoked only from the crucial areas for regulating erection. Schmidt et al. reported that ibotenic acid lesions of LPOA disrupted penile erection during REM sleep, while leaving waking-condition erection intact, and suggested that the LPOA plays a crucial role in penile erection during REM sleep (Schmidt et al., 2000). Our finding that penile erections were evoked by
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stimulation of the LPOA further confirms that the neural substrate for penile erection is located in the LPOA. Schmidt et al. (2001) have also reported that injections of carbachol in the LPOA induce penile erections. We found that stimulation of the laterodorsal tegmental nucleus (LDT, one of the brainstem cholinergic nuclei) induced erection (Salas et al., 2007), and suggested that the LDT is a cholinergic source for induction of erection in the LPOA. The MPOA is known to play crucial roles in the regulation of reproductive 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 in penile erection. Schmidt et al. (2000) found that MPOA lesions had almost no effect on waking-state or REM sleep-related erection. Liu et al., (1997a) have reported that male rats with MPOA lesions exhibit severe deficits in copulation but little or no decrement in non-contact erection (NCE), while BST lesions cause impairment of NCE with only slight deficits in copulation, suggesting that the BST plays an important role in mediating NCE. In the present study, although erections were evoked from the MPOA, the stimulus intensities required for them were higher than those for elicitation of erection from the LPOA or BST. The MPOA thus appears to play a role in penile erection different from those of the LPOA and BST. Considering the spread of stimulus current (Fig. 2), it is possible that the stimulation of higher intensity applied to the MPOA invaded the LPOA or BST. The neurons in the BST send projections to the LPOA and MPOA (Dong and Swanson, 2006). Liu et al. (1997a) have suggested that an extra-MPOA pathway mediates NCE. The system extending from BST to LPOA might correspond to this pathway. 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), while lesions of PVN disrupts NCE (Liu et al., 1997b; Melis et al., 2001). The PVN projects directly to the intermediolateral region of the spinal cord (Ranson et al., 1998). In the present study, erection was evoked by PVN stimulation with lower intensities than the MPOA (Fig. 6A) and higher probability than any other brain areas (Table 2). These facts support the idea that PVN neurons receiving dopaminergic or glutamatergic inputs and projecting directly to the spinal cord act as the final output of the erection regulating system in the hypothalamus.
4.
Conclusion
In unanesthetized rats, penile erection was evoked by the electrical stimulation of the LPOA, MPOA, BST, PVN RE and LS. Among them, lower-intensity stimulus was effective in the LPOA, BST, PVN and RE, but not in the MPOA. In anesthetized condition, the effective areas were restricted to the LPOA, MPOA and BST. The present results suggest the difference of the lateral and medial subdivisions of the POA, emphasizing the role of the lateral subdivisions (LPOA and BST), in the regulation 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 a 12:12-h light/dark cycle at an ambient temperature of 22.0 ± 0.1 °C with food and water available ad libitum. Of them, 13 were used under urethane anesthesia, while 7 were used unanesthetized in head-restrained condition. All procedures were carried out under the control of the Animal Research Committee in accordance with the Guidelines for Animal Experiments of Fukushima Medical University and the Japanese Animal Protection and Management Law (No. 105). All efforts were made to minimize the number of animals used and their suffering.
5.1.1.
Surgical procedure
For acute experiments, animals were anesthetized with urethane (1.2 g/ kg, i.p.). For surgery for chronic experiments, sodium pentobarbital (50 mg/kg) was intraperitoneally injected and additional doses were given to maintain the level of anesthesia. As previously described, the pressure of the corpus spongiosum of the penis (CSP) was measured telemetrically (Gulia et al., 2008; Nout et al., 2007; Schmidt et al., 1994; Salas et al., 2007). In brief, the distal portion of the bulb of the corpus spongiosum of the penis (CSP) was gently exposed and the catheter tip of the telemetric transducer (TA11PA-C40, Data Sciences International, St. Paul MN) was inserted into the bulb through a slit made by a needle. The body of the transducer was fixed subcutaneously to the abdominal muscle. The catheter was secured using a biological glue (Vetbond, 3 M Animal Care Products) at the point of entrance of the catheter and was sutured to the fascia overlying the shaft of the penis. To record the electromyogram (EMG) of the bulbospongiosus (BS) muscle, a pair of stainless wires were inserted into the BS muscle and passed subcutaneously to an incision of the skin over the skull which had been made in advance of the implantation. To assess vigilance state, stainless steel screws (tip diameter= 1.4 mm) for recording of the cortical electroencephalogram (EEG) were implanted in the skull overlying the frontal and parietal cortices and, in unanesthetized, head-restrained rats, wire electrodes were implanted in the neck muscle to record the electromyogram (EMG). For painless fixation of the unanesthetized rats to a stereotaxic frame during experiments, a U-shaped plastic plate (width 20 mm, length 30 mm, thickness 5 mm) was attached to the 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 rats were given a 1-week period for recovery.
5.2.
Experimental procedures
One day before the experiment, rats were attached to the stereotaxic frame under ketamine anesthesia (50 mg/kg) using the U-shaped plate, and a small hole was made through the occipital bone using a dental drill to expose the surface of the cerebral cortex above the preoptic area. The rats were deprived of sleep for 12 h in a slowly rotating wheel (diameter 37 mm, width 10 cm, 1.2 rev/min) with free access to food and water. On
the day of the experiment, rats were returned to their cage to rest for 2–3 h. They were then fixed to the stereotaxic frame using the U-shaped plate. The stimulation electrode consisted of a glass pipette which had a carbon fiber (diameter, <10 μm; resistance, 4–6 MΩ) protruding from the tip about 20–30 μm and a cavity filled with Woods metal (Takakusaki et al., 1989). To determine sites most effective for induction of erection, stimulation was applied, usually at 0.2 mm intervals, by moving the electrode with an oil microdrive manipulator. Stimuli were given 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 leg movement. Therefore, in most cases the stimuli were limited to less than 100 μA. The most effective site along the track was marked by passing 20 μA positive current (DC) for 30 s. After completion of the experiment, the rats were deeply anesthetized with pentobarbital and perfused transcardially with 300 ml of physiological saline followed by 300 ml of 10% formalin. The brain was then removed, postfixed in the same fixative, soaked in 30% sucrose and sectioned in the coronal plane at a thickness of 50 μm. The sections were stained with neutral red. Stimulation sites were identified under light microscopy using standard nomenclature according to the rat brain atlas of Paxinos and Watson (1997).
5.2.1.
Definition of responses
As shown in Fig. 1, erection is composed of a slow CSP pressure increase and sharp CSP pressure peaks concurrent with bursting BS muscle activity. The slow CSP pressure increase reflects the flow of blood into the CSP caused by the distention of the blood vessels supplying the CSP, and is termed the vascular component, while the sharp peaks reflect each of the erectile events of the penis (flip), which are caused by bursting discharges of the BS muscles (muscular component). In the rat, the vascular component is associated with an increase in the baseline erectile tissue pressure from approximately 10–15 mmHg in the flaccid state (Flaccid Baseline, FB) to a tumescence pressure up to approximately 50–70 mmHg. The muscular component is easily identified by the sharp, suprasystolic CSP pressure peaks occurring on top of the tumescence pressure. Normal erection was defined as a tumescence pressure at least 30 mmHg higher than the FB. The start of erection was defined as the time the CSP pressure reached FB+ 30 mmHg, while the end of erection was defined as the time when CSP pressure fell below FB+ 30 mmHg for more than 15 s. To distinguish between two erections occurring in close temporal association, a new erection was considered to occur when pressure dropped below FB + 30 mmHg for more than 15 seconds or when it dropped below FB+ 15 mmHg for more than 5 seconds. For statistical purposes, CSP pressure peaks greater than FB + 80 mmHg were evaluated. Vascular latency was measured as the time from the start of the stimulus to the start of erection. Peak frequencies were obtained by dividing the number of peaks by the time from the first peak to the last peak. Data were analyzed using Spike 2 data software (Cambridge Electronic Design, UK).
5.2.2.
Data analysis
The Mann–Whitney U test with Bonferroni correction was performed to compare the medians of response values among
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response types (Table 1) or among stimulation sites. The χ2 test was performed to compare the probability of each type of response between anesthetized and unanesthetized groups. Statistica software was used for these analyses. Differences were considered significant at p < 0.05. Because the maximum value measured by the transducer was 400 mmHg, the CSP peaks sometimes exceeded this value. A CSP pressure > 400 mmHg was counted as 400 mmHg for purposes of statistical analysis.
Acknowledgments This study was supported by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science to Y. Koyama. The authors thank Ms. Nobuko Anzai for her technical assistance.
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