To investigate the role of serotonin (5-HT) receptor 1A or 7 in regulating lordosis behavior in female rats, ovariectomized rats were treated with 3 kinds of receptor agonists and lordosis behavior was observed. The injected agents were the selective 5-HT1A receptor agonist, buspirone (BUS), the highly selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin ((±)8-OH-DPAT), and the 5-HT1A and 5-HT7 receptor agonist, (R)-8-hydroxy-2-(di-n-propylamino)tetralin (( )8-OH-DPAT). A behavioral test was performed after ovariectomy and subcutaneous implantation of a silicon tube containing estradiol. Female rats in which the lordosis quotient (LQ) was over 70 were intraperitoneally injected with several doses of these agents. As a result, in the BUS group, the dose of 3 mg/kg bw, but not 1 mg/kg was effective for suppressing lordosis. On the other hand, an inhibitory effect was observed from 0.25 mg/kg and 0.5 mg/kg in the ( )8-OH-DPAT and (±)8-OH-DPAT groups, respectively. In the time-course experiment, in all drug-treated groups, LQ decreased to lower than 20 after 15 min and low LQ continued for 1 hr at least. Measurement of locomotor activity using an infrared sensor system showed no relation between the decrease in lordosis by these agents and spontaneous locomotion. These results indicate that 5-HT1A is strongly involved in the lordosis-inhibiting circuit of the serotonin neurons.
Highly estrous female rats show lordosis and soliciting behaviors with male mounting. These characteristic sexual behaviors are regulated by facilitatory and inhibitory systems in the central nervous system. The ventromedial hypothalamic nucleus (VMH) exerts an estrogen-dependent facilitatory influence in regulating lordosis (Pfaff et al., 1994). On the contrary, the lateral septum (LS) and the preoptic area (POA) exert a lordosis-inhibitory influence (Yamanouchi, 1997). These facilitatory and inhibitory mechanisms in the forebrain are modified by the limbic system and the lower brainstem.
In the midbrain, the dorsal raphe nucleus (DRN) plays an important role in inhibiting lordosis behavior, because destruction or ventral cutting of this nucleus facilitated lordosis behavior in female rats (Yamanouchi and Arai, 1985; Kakeyama and Yamanouchi, 1996; Kakeyama et al., 1997). Electrical stimulation of the DRN inhibited lordosis in female rats (Arendash and Gorski, 1983). Furthermore, since a DRN lesion causes male rats to show lordosis, the quantity or quality of the inhibition of the DRN leads to sex differences in the brain (Kakeyama and Yamanouchi, 1992).
The DRN contains a large number of serotonergic neurons (Törk, 1985). Since lordosis is enhanced by treatment with the serotonin (5-HT) synthesis inhibitor, p-chlorophenylalanine (PCPA) (Zemlan et al., 1973), but is not enhanced in rats with a DRN lesion (Kakeyama and Yamanouchi, 1993), it appears that serotonergic neurons in the DRN play an inhibitory role in lordosis-regulation. The axons of serotonergic neurons in the DRN extend to the lordosis regulating mechanisms in the hypothalamus, including the VMH and the POA (Parent et al., 1981; Törk, 1985). Among the 5-HT receptors, 7 families (from 1 to 7) have been classified (Hoyer et al., 1994). In the hypothalamus 5-HT1A, 5-HT2c and 5-HT7 receptors have been found (Hoyer et al., 1994; Barnes and Sharp, 1999).
It has been suggested that the 5-HT1A receptor is involved in the lordosis inhibiting mechanism, because 5-HT1A receptor agonists, (±)8-OH-DPAT (Mendelson and Gorzalka, 1986b) or buspirone (Mendelson and Gorzalka, 1986a) decreased lordosis. In contrast, since lordosis is facilitated by treatment with the 5-HT2A/2C receptor agonist, DOI, and conversely, is inhibited by the antagonist, ketanserin (Wolf et al., 1998; Wolf et al., 1999), 5-HT2A/2C receptor plays an important role in the lordosis facilitating mechanism. Moreover, treatment with the 5-HT3 receptor antagonist, tropisetron, decreased lordosis (Maswood et al., 1997; Maswood et al., 1998). In this study, to clarify the role of 5-HT1A and 7 receptors in regulating lordosis, a comparative analysis was performed using the highly selective 5-HT1A receptor agonist, (±)8-OH-DPAT, 5-HT1A and 5-HT7 receptor agonist, (+)8-OH-DPAT, or relatively selective 5-HT1A receptor agonist, buspirone.
MATERIALS AND METHODS
Female Wistar rats (215–295 g) were purchased from Takasugi Animal Farm (Saitama, Japan). The rats were housed under conditions of controlled temperature (23–25°C) and photoperiod (14 : 10, light : dark). Food and water were available ad libitum. The present experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals in the Human Science Department of Waseda University. All female rats were ovariectomized under ether anesthesia. Rats were implanted with a silicon tube (inner diameter, 1.57 mm; outer diameter, 3.18 mm; length, 30 mm; Kaneka Medix Co., Osaka, Japan) containing estradiol-17β (E2; Sigma Chemical Co., St. Louis, MO, USA) subcutaneously within one week after ovariectomy. Three experiments were then performed.
In the 1st experiment, the effect of 3 kinds of 5-HT receptor agonist on lordosis behavior was investigated. Six days after the implantation of E2, the first behavioral test was carried out. In the behavioral test, each animal was placed in a plastic observation cage (40×60×50 cm) with two vigorous male rats. The lordosis quotient (LQ; number of lordosis reflex / 10 mounts ×100) was recorded.
One hr after the 1st behavioral test, rats in which the LQ was over 70 in the 1st test were injected with 3 kinds of 5-HT receptor agonist and the 2nd behavioral test was performed. Eight rats in each group were injected with 1, 3, or 5 mg/kg bw buspirone (buspirone hydrochloride; BUS; 1 mg/ml saline; Sigma) intraperitoneally. In 7 rats of each group, the animals were injected with 0.25, 0.5 or 1 mg/kg 8-hydroxy-2-(di-n-propylamino)tetralin ((±)8-OH-DPAT; Sigma), or 0.25, 0.5 or 1 mg/kg (R)-8-hydroxy-2-(di-n-propylamino)tetralin ((+)8-OH-DPAT; Sigma) intraperitoneally. Fifteen females were injected with 1 ml/kg saline as the control group. Thirty minutes after injection, the second behavioral test was started.
In the 2nd experiment, to examine the time-course change of LQs after injection of the agonists, 1 hr after the 1st behavioral test, female rats were injected with 3 mg/kg BUS (n=6), 1 mg /kg (±)8-OH-DPAT (n=5), 1 mg/kg (+)8-OH-DPAT (n=5) or 1 ml/kg saline (n=10) and behavioral tests were performed at 15, 30, 60, and 120 min after injection.
In the 3rd experiment, to check the effect of 5-HT receptor agonists on locomotor activity, E2-treated ovariectomized rats were injected with 3 mg/kg BUS (n=5), 1 mg /kg (±)8-OH-DPAT (n=5), 1 mg/kg (+)8-OH-DPAT (n=6) or 1 ml/kg saline (n=10) and just after injection, measurement of spontaneous locomotor activity was started. The regime of treatments with estrogen and 5-HT agonists was the same as those in the 1st experiment. Spontaneous loco-motor activity was measured continuously for 3 hr from 14:00 by means of an infrared sensor system (SUPERMEX; MuromachiKikai, Tokyo, Japan). The measured locomotor activities were totaled every 15 min in each rat.
The differences in the mean LQ between before and after injection in each group were analyzed by Wilcoxon signed rank test. The differences in the mean LQ among groups were analyzed by the Kruskal-Wallis test and followed by the Mann-Whitney U-test. The differences in the mean LQ in the time-course test and loco-motor activity among the groups were analyzed by analysis of variance (ANOVA) of repeated measures. The differences in the loco-motor activities among the groups at each time were analyzed by t-test.
The mean LQs in the saline- and BUS-treated groups are shown in Fig. 1. In the saline-treated control group, the mean LQs were high both before and after injection. In the 1 mg/kg BUS-treated group, the mean LQ after injection was almost the same as that in the control group or before injection. In contrast, the mean LQs in the 3 and 5 mg BUS-treated groups were lower than those before injection (P<0.05) or in the control group and 1 mg group (P<0.0001) after injection.
The mean LQs in the (±) or (+)8-OH-DPAT-treated group are shown in Fig. 2. In both the (±) and (+)8-OHDPAT-treated groups, after treatment at the doses of 0.5 or 1 mg/kg, the mean LQs were lower than those before injection (P<0.05). Furthermore, in the 0.25 mg/kg (+)8-OHDPAT-treated group, the mean LQ was lower than that before injection (P<0.05). A comparison between the (±) and (+)8-OH-DPAT-treated groups revealed the absence of any significant differences in the mean LQ of each dose.
Time courses of the inhibition of the lordosis response by 5-HT1A receptor agonists are shown in Fig. 3. High LQ was sustained after injection with saline during a period of 3 hr. In all the drug-treated groups, the mean LQs decreased to lower than 20 after 15 min, and low level of LQs continued for 1 hr. At 2 hr after injection, the mean LQs recovered to higher than 60. At each time, there was significant difference in the mean LQ between the control and drug-treated groups (P<0.0001).
Locomotor activity immediately after injection was high, but decreased soon thereafter and the level was sustained for 3 hr with small changes in the saline-treated control group (Fig. 4). In the 1 mg/kg (±)8-OH-DPAT-treated group, similar locomotor activity to that of the control was observed during a period of 3 hr. In contrast, (+)8-OH-DPAT-treated rats showed higher locomotor activity than that of the control during a 1-hr period, but then decreased to the same level of the control. In the BUS-treated group, the first 15-min locomotor activity was lower than those of other groups (P<0.001 vs. saline, P<0.01 vs. (±)8-OH-DPAT, P<0.0001 vs. (+)8-OH-DPAT).
In the present experiment, the dose of 3 mg/kg bw, but not 1 mg/kg BUS inhibited lordosis behavior in female rats. On the other hand, in the (±)8-OH-DPAT-treated group, 0.5 mg/kg was sufficient to suppress lordosis. Furthermore, (+)8-OH-DPAT was most effective, because the dose of 0.25 mg/kg inhibited lordosis behavior. These results are in agreement with the reports that 8-OH-DPAT has a strong influence on the inhibition of lordosis, compared with BUS (Smith and Peroutka, 1986; Uphouse et al., 1992).
BUS has been reported to act on 5-HT1A, dopamine (DA) or noradrenaline (NA) receptors (Trulson and Henderson, 1984; Peroutka, 1985; Reader et al., 2000). It has been reported that DA receptors contribute to the stimulatory mechanism of lordosis behavior, because direct application of DA agonist into the brain was found to enhance lordosis (Foreman and Moss, 1978). The NA neuron is also involved in the lordosis facilitating mechanism (Crowley et al., 1978; Etgen, 1990). Therefore, stimulation of monoaminergic receptors other than 5-HT receptor may rather weaken the lordosis-inhibiting effect of BUS.
(±)8-OH-DPAT acts on 5-HT1A receptor specifically in the brain (Uphouse et al., 1992; Uphouse and Caldarola-Pastuszka, 1993). Thus, (±)8-OH-DPAT and BUS are thought to act on 5-HT1A receptors and inhibit lordosis behavior by binding to 5-HT1A receptor in the female rat brain.
On the other hand, (+)8-OH-DPAT has been reported to act on 5-HT1A and 5-HT7 receptors (Cornfield et al., 1991; Barnes and Sharp, 1999). Some 5-HT receptors have been reported to play a facilitative role in regulating lordosis behavior, because lordosis is enhanced by treatment with a 5-HT2A/2C receptor agonist (Wolf et al., 1998; Wolf et al., 1999) and is suppressed by a 5-HT2A/2C antagonist (Wolf et al., 1998; Wolf et al., 1999) or a 5-HT3 receptor antagonist (Maswood et al., 1997; Maswood et al., 1998). If 5-HT7 receptor is involved in the lordosis-facilitating mechanisms of the serotonergic neuron, lordosis is expected to increase by treatment with (+)8-OH-DPAT. In the present experiment, rather, (+)8-OH-DPAT was more effective in suppressing lordosis than was (±)8-OH-DPAT. This suggests that similar to 5-HT1A receptor, 5-HT7 receptor is concerned with the lordosis-inhibiting system. However, there was no statistical difference in the mean LQ between the (+)8-OH-DPAT group and the (±)8-OH-DPAT group in all tests. Further experiments using a more selective 5-HT7 receptor agonist than (+)8-OH-DPAT or direct application of the drug to the candidate portion in the brain are necessary.
In all of the drug-treated groups, LQs decreased 15 min after injection and the low level of LQs continued for 1 hr. LQs recovered to some extent after 2 hr. These results are in agreement with the report that a decreased LQ in female rats injected with BUS or 8-OH-DPAT into the VMH recovered in about 2 hr (Uphouse et al., 1992). Yu and Lewander (1997) reported that 15 min after subcutaneous injection of (+)8-OH-DPAT, this agonist had been found in the brain. This supports our finding of an acute inhibitory effect of these drugs on lordosis. There were no differences in the locomotor activities among the groups with some exceptions in the early period after injection in the (+)8-OH-DPAT and BUS groups. However, because the lordosis inhibiting effect was observed in all groups regardless of the locomotor condition, the effects of these agonists are thought to act on the lordosis inhibiting system without affecting the regulatory center of locomotion and decreased lordosis.
Direct hypothalamic application of 5-HT1A receptor agonist has been reported to suppress lordosis in female rats (Uphouse et al., 1992). In the DRN, axons of serotonergic neurons, which exert a lordosis-inhibiting influence (Kakeyama and Yamanouchi, 1992), extended to the hypothalamus, the POA and the VMH (Parent et al., 1981; Törk, 1985). After a lesion formed in the DRN, the contents of serotonin in the POA and the VMH decreased in female and male rats (Kakeyama et al., 2002). In the POA and the VMH, the amount of serotonin decreased when female rats exhibited lordosis behavior (Luine, 1993; Gonzalez et al., 1997). 5-HT1A receptors exist in the POA and the VMH (Gonzalez et al., 1997). From these results, the lordosis modulating functions of the POA and the VMH received an inhibiting influence from the DRN serotonergic neurons through the 5-HT1A receptor system.
5-HT1A receptor function is influenced by estrogen (Jackson and Uphouse, 1998). On the other hand, estrogen does not seem to have a direct effect on the lordosis regulating function in the DRN, because direct application of the crystal estrogen into the DRN did not facilitate lordosis in subthreshold doses of estrogen-treated ovariectomized rats (Satou and Yamanouchi, 1999). Furthermore, it has been reported that the number of serotonin-immunopositive cells in the DRN is not influenced by estrogen (Lu et al., 2001). These reports suggest that regulation of 5-HT1A receptors by estrogen may be via neurons other than the serotonergic neurons of the DRN. Further experiments are needed to clarify the relationship between 5-HT1A receptor-modulation and sex steroid hormone.
This study was supported by a Grant-in-Aid from the Ministry of Education, Science, Culture and Sports of Japan (14540618), grants for special research project (2003A-605) and AR Center for Human Sciences of Waseda University to K.Y.