Pharmacological Studies on the Role of 5-HT1A Receptors in Male Sexual Behavior of Wildtype and Serotonin Transporter Knockout Rats

Brain serotonin (5-HT) neurotransmission plays an important role in male sexual behavior and it is well established that activating 5-HT1A receptors in rats facilitate ejaculatory behavior. However, the relative contribution of 5-HT1A somatodendritic autoreceptors and heteroreceptors in this pro-sexual behavior is unclear. Moreover, it is unclear whether the contribution of somatodendritic 5-HT1A autoreceptors and postsynaptic 5-HT1A heteroreceptors alter when extracellular 5-HT levels are chronically increased. Serotonin transporter knockout (SERT–/–) rats exhibit enhanced extracellular 5-HT levels and desensitized 5-HT1A receptors. These rats model neurochemical changes underlying chronic SSRI-induced sexual dysfunction. We want to determine the role of presynaptic versus postsynaptic 5-HT1A receptors in the pro-sexual effects of 5-HT1A receptor agonists in SERT+/+ and in SERT–/– rats. Therefore, acute effects of the biased 5-HT1A receptor agonists F-13714, a preferential 5-HT1A autoreceptor agonist, or F-15599, a preferential 5-HT1A heteroreceptor agonist, and S15535 a mixed 5-HT1A autoreceptor agonist/heteroreceptor antagonist, on male sexual behavior were assessed. A clear and stable genotype effect was found after training where SERT+/+ performed sexual behavior at a higher level than SERT–/– rats. Both F-15599 and F-13714 induced pro-sexual activity in SERT+/+ and SERT–/– animals. Compared to SERT+/+, the F13714-dose-response curve in SERT–/– rats was shifted to the right. SERT+/+ and SERT–/– rats responded similar to F15599. Within both SERT+/+ and SERT–/– rats the potency of F-13714 was much stronger compared to F-15599. S15535 had no effect on sexual behavior in either genotype. In SERT+/+ and SERT–/– rats that were selected on comparable low sexual activity (SERT+/+ 3 or less ejaculations and SERT–/– 5 or less ejaculations in 10 weeks) S15535 also did not influence sexual behavior. The two biased compounds with differential effects on 5-HT1A auto- and hetero-receptors, exerted pro-sexual activity in both SERT+/+ and SERT–/– rats. Applying these specific pharmacological tools has not solved whether pre- or post-synaptic 5-HT1A receptors are involved in pro-sexual activity. Moreover, the inactivity of S15535 in male sexual behavior in either genotype was unexpected. The question is whether the in vivo pharmacological profile of the different 5-HT1A receptor ligands used, is sufficient to differentiate pre- and/or post-synaptic 5-HT1A receptor contributions in male rat sexual behavior.

As mentioned before, chronic SSRI treatment results in enhanced 5-HT levels often causing sexual dysfunctions (Segraves and Balon, 2014). The exact mechanisms for these dysfunctions remain unclear, but are high likely due to alterations in the 5-HT 1A receptor. Male rats lacking the serotonin transporter (SERT −/− ) display a robust genotype that has a lower basal ejaculatory performance than wildtype rats (SERT +/+ ) or heterozygous serotonin transporter knockout (SERT +/− ) rats Esquivel-Franco et al., 2018). More specific, due to the lack of the serotonin transporter SERT −/− rats have a nine-fold increase in extracellular 5-HT levels (Homberg et al., 2007), decreased number of ejaculations and an increased ejaculation latency  compared to SERT +/+ rats. This genetic animal model has therefore been proposed and used as an animal model of spontaneous or SSRIinduced delayed ejaculation in humans. Chronic SSRI use in men may result in several side-effects including increased ejaculation threshold, resulting in a delayed ejaculation latency or sometimes even absent ejaculation, associated with a reduction in sexual desire (Waldinger et al., 1998;Hirschfeld, 2003;Balon, 2006;Rubio-Casillas et al., 2015). This is believed to be caused by the combination of enhanced 5-HT levels and diminished 5-HT 1A receptor functioning (both pre-and post-synaptic) similar to chronic SSRI-treatment in normal animals , or short acting SSRIs like dapoxetine in fast ejaculating rats (Clément et al., 2012). Although conflicting findings on the effects of acute and chronic SSRI treatment have been reported, Clément et al. (2012) mention this is explained by distinct pharmacokinetics rather than pharmacodynamic properties as dapoxetine has rapid peak plasma concentrations which delays ejaculation frequencies in men with premature ejaculation. In rats the dapoxetine profile is less clear, although it is suggested by the authors that in faster ejaculating rats dapoxetine seems to delay the ejaculation latency (Clément et al., 2012). In particular this 5-HT 1A receptor desensitization phenomenon is relevant here to further provide more clarity as to the potency of the biased agonists to stimulate sexual behavior. SERT −/− rats have higher extracellular serotonin levels than SERT +/+ animals which is comparable to levels after chronic SSRI administration (Homberg et al., 2007). Pharmacological experiments in these rats indicated that rats lacking the SERT have altered 5-HT 1A receptor reactivity; the altered 5-HT 1A receptor functioning is probably not a global phenomenon, but might be limited to some specific subpopulations of 5-HT 1A receptors (not necessarily preor post-synaptic), as indicated by changed autonomic responses like core body temperature in SERT +/+ and SERT −/− animals. The 5-HT 1A receptor population involved with hypothermia was not sensitive, while the 5-HT 1A receptor population involved with hyperthermia was still sensitive Olivier et al., 2008). Experiments performed in male sexual behavior  also indicated that likely at least two populations of 5-HT 1A receptors are involved in its expression. However, it is worthwhile to mention that 5-HT 1A receptors can co-localize with 5-HT 7 receptors in the cell-membrane (Renner et al., 2012). It has been postulated that heterodimerization of these receptors may influence the desensitization of 5-HT 1A autoreceptors caused by SSRIs (Naumenko et al., 2014). In addition, 5-HT 1A receptors are G-protein coupled receptors activating different intracellular signaling pathways, which are brain region specific. Activation of different G-protein cascades may therefore play a role in the activation of 5-HT 1A receptors in specific cellular environments, while having no effect on other subpopulations of the same receptor (Newman-Tancredi, 2011). For performing sexual behavior, activation of one population of 5-HT 1A receptors is needed and we postulated that this pool is desensitized in SERT −/− rats. The pro-sexual effects of 8-OH-DPAT are probably mediated via 5-HT 1A receptors, which are not changed or somewhat less sensitive in SERT −/− rats. This difference makes the SERT −/− rat a further attractive model to test the different 5-HT 1A receptor-modulating drugs, F15599, F13714 and S-15355, as it may provide information on the adaptation of pre-and post-synaptic 5-HT 1A receptors due to chronic high 5-HT levels, which may aid in the treatment of sexual dysfunction caused by SSRI treatment.
Finally, we selected male rats that, after extensive training (Pattij et al., 2005), display a low level of sexual behavior, i.e., low number of ejaculations. Because 5-HT 1A receptor agonists facilitate ejaculation, a too high initial level of the number of ejaculations would probably interact with the prosexual effects of these drugs. The purpose of this study was to use functionally selective agonists for either preor post-synaptic 5-HT 1A receptors to identify the role of somatodendritic (auto) receptors and post (hetero) receptors in sexual behavior. A second goal was to use SERT −/− rats because they model SSRI-induced delayed ejaculation in humans, and hence may provide insight in the adaptation of specific 5-HT 1A hetero-or auto receptors due to chronic increased extracellular 5-HT levels. Thus, we investigated whether chronic exposure to high 5-HT levels affected the prosexual effects of 5-HT 1A agonists, and whether pre-or postsynaptic receptors were differently affected in SERT −/− rats compared to SERT +/+ rats. We used F13714, F15599, and S15535 in normal (SERT +/+ ) and SERT −/− rats, and hypothesized that these drugs would have differential effects on sexual behavior and that SERT −/− rats would display desensitized response to these drugs.

Animals
Wistar rats were bred in our animal facility (University of Groningen, GELIFES) using serotonin transporter (SERT) heterozygous males and females, resulting in male and female SERT wild type (SERT +/+ ), heterozygous (SERT ± ) and homozygous or knock out (SERT −/− ) rats. On postnatal day 21 pups were weaned and ears were punched for individual recognition and genotyped as reported previously (El Aidy et al., 2017). We used two groups of animals, the first one (normal ejaculating rats) consisting of sixty-three male SERT (SERT +/+ , n = 32), and (SERT −/− , n = 31) rats and the second one (slow ejaculating rats) of 32 male (16 SERT +/+ and 16 SERT −/− ) rats, all of them of at least 12 weeks old when used for sexual behavior experiments.
Female SERT ± and SERT +/+ were used as sexual stimulus females (n = 120) as SERT +/+ and SERT ± rats do not differ in basal sexual activity (Snoeren et al., 2010). Rats were housed under reversed dark-light conditions (12 h light:12 h dark, lights off from 8:00 AM to 8 PM). After 6-weekly training tests (30 min/test), male rats were considered sexually trained and classified based on ejaculation frequencies per test. Male rats display, after extensive training, a rather stable sexual phenotype (Pattij et al., 2005;Olivier et al., 2006;Chan et al., 2008). In these experiments, for the normal ejaculating 24 rats were selected (from 14 different dams, a maximum of 3 SERT +/+ and/or 3 SERT −/− rats were used from the same litter) that showed a normal ejaculatory phenotype (between 1 and 2 ejaculations per test after training, for the last three sessions) and for the slow ejaculating rats 20 (from 8 dams, a maximum of 5 SERT +/+ and/or 2 SERT −/− rats were used from the same litter) that showed a rather low sexual phenotype (between 0 and 1 ejaculation per test after training, for the last three sessions) were selected. We summed all ejaculations per rat for all training weeks in Supplementary Figure 1 (group 1) and 2 (group 2). The most left tail-side of the distribution was selected. Animals were socially housed (2-5 per cage, maximum 4 for males). Cages were enriched with wooden gnawing blocks and nesting material (Envirodri).
Thus, to select normal ejaculating rats 32 SERT +/+ and 31 SERT −/− rats were sexually trained for 6 weeks and a total of 12 SERT +/+ and 12 SERT −/− rats were selected with a normal average number of ejaculations. For selection of the slow ejaculating rats 16 SERT +/+ and 16 SERT −/− rats were sexually trained for 6 weeks and a total of 10 SERT +/+ and 10 SERT −/− rats were selected with a normal and low average number of ejaculations (because this enhances the sensitivity of the anticipated improvement in sexual behavior by the 5-HT 1A compounds and to match the control group as much as possible to the knock-out animals). Experiments in the normal ejaculating rats lasted 13 weeks in total (after training), and 4 weeks in total (after training) for sow ejaculating rats. Animals were used only once a week to guarantee sufficient drug washout time. Rats had ad libitum access to food and water. This study was carried out in accordance with the principles of the EU Directive 2010/63/EU. All efforts were made to minimize the number of animals and their suffering.

Female Rats
Female stimulus rats were tubal ligated in order to prevent pregnancies. To perform tubal ligation surgery, females were anesthetized (Isoflurane) and given pain relief (Fynadine, 0.1 mg/100 g) before surgery, and 24 and 48 h after surgery. Females were at least 12 weeks old when surgery was performed, and 2 weeks of recovery were given before they were made intentionally receptive with estradiol (50 µg in 0.1 ml oil, S.C., 36-48 h before the test) before the sexual behavior training tests and experiments. Females were used not more than once in 2 weeks and not more than two times per experimental day.

Drug Treatment and Behavioral Experiments
For the first experiment in normal ejaculating rats, animals received all dosages of F13714 and F15599 in a crossoverrandomized design in order to prevent order effects; after this experiment, S15535 was administered in a randomized design. For the second experiment in slow ejaculating rats, animals were only administered S15535 in a randomized design similar to the first set of animals. As described previously in Olivier et al. (2017), when pharmacological tests are performed, male rats are given a 30-min habituation time in the test boxes right after drug administration via IP injection, before the female rat is introduced. All behavior during the 30-min test is videorecorded after introduction of the female and were also livescored; the following parameters of the ejaculation series were deduced : number of ejaculations/test (E), number of mounts (M), number of intromissions (I), latency (s) to first mount (ML), latency (s) to first intromission (IL) and latency (s) to the first ejaculation (EL). After ejaculation, the post ejaculatory latency (PEL(s)) was calculated, using the time from the first ejaculation and the time of the first mount/intromission (whatever occurred first) of the second ejaculation series. Intromission Ratio (IR) was calculated as: IR = (#I/(#I + #M)) × 100%. EL was calculated using the time of the EL from the first ejaculation series minus the intromission latency of the first ejaculation series (EL = EL -IL). These parameters were used to run the statistical analysis.
Because it is important to have comparable pharmacodynamics and kinetics in pharmacological studies, a test of fixed duration has been chosen: 30 min (1800 s). In the cases where drug-treatment had no "effect" on ejaculation and sexual behavior, or few or no animal achieved a first ejaculation it was not possible to perform statistical analyses and for those cases we assigned values of 1800 s (i.e., the maximum test duration) for some latencies (ejaculation, mount and intromission latency), although this is undoubtedly a matter of discussion as we have discussed before Olivier et al., 2017). All tables and figures show the results for the first Ejaculation Series only.

Drugs
F15599 and F13714 (Pierre Fabre Pharmaceuticals, France; Lot # SBR1401003 and # JLM3001201, resp.) and S-15535 (Servier Pharmaceuticals, France; Lot B01JLP061A) were dissolved in NaCl 0.9% (saline) and each solution was freshly prepared on each testing day. All drugs were administered via intraperitoneal (IP) injection 30 min before the test.
Training (Table 1) For the normal ejaculating group, rats were sexually trained for 6 times (30 min, once a week). For the slow ejaculating group, rats were sexually trained 10 times (30 min, once a week). The latter animals received extra training due to the extreme low sexual performance to assess and stabilize their basal sexual activity. Rats habituated for 10 min to the testing box right before the training session. After the habituation period a receptive female was introduced in the box and sexual behavior was assessed for 30 min. Non-receptive females were switched for a different receptive female. The training and testing occurred in wooden rectangular (57 cm × 82 cm × 39 cm; glass wall) testing boxes filled with regular bedding material. To stimulate sexual behavior, bedding material was not changed during the training and testing to preserve pheromones of previous rounds and to create a more competitive sexual environment.
Only males showing stable normal (1-2 ejaculations, for experiments 1 and 2 and low (0-1 ejaculations for experiment 3) ejaculation levels in the last three tests were used in the Twenty-four normal ejaculating male rats were selected (N = 12 per SERT genotype) and were tested in a crossover design. Rats received vehicle (saline), 0.01, 0.04, 0.16, and 0.64-mg/kg F15599 and 0.0025, 0.01, 0.04, and 0.16-mg/kg F13714 via intraperitoneal (IP) administration. Experiments were performed once per week on the same testing day, over 9 weeks and animals and treatment were randomized over the 9 weeks. Although the experiments with these two drugs were performed together, we performed the statistical analysis separately for each compound.

Experiment 2 (Normal Ejaculating Rats): S15535 Dose Response
The same 24 animals from experiment one received vehicle (saline), 0.25, 1 and 4-mg/kg S15535, IP in a randomized design. Testing was performed over 4 weeks and always on the same day per week.
Experiment 3 (Slow Ejaculating Rats): S15535 Dose Response 10 SERT +/+ and 10 SERT −/− rats were selected for low numbers of ejaculation. Rats received vehicle (saline), 0.25, 1, and 4-mg/kg S15535, via IP administration in a randomized design. Testing was performed over 4 weeks and always on the same day per week.

Statistical Analyses
Differences in baseline ejaculation numbers during the training between genotypes were analyzed using two-way ANOVA for repeated measures, with genotype as between-and time (weeks) as within-subjects factors. Where appropriate, an independent t-test was performed. For the F19955, F13714, and S15535 doseresponse experiments, a two-way ANOVA for repeated measures was performed with dose as within-subject factor (5 levels) and genotype as between-subject factor (2 levels). Where appropriate one way-ANOVA with LSD post hoc was performed. All statistical analyses were performed using the Statistical Package for Social Sciences for Windows version 25 (LEAD technologies, Chicago, United States). Level of significance was set at p < 0.05.

Sexual Stability
The sexual performance of the selected experimental animal groups that exhibited a normal (1-2 ejaculations) and a low basal ejaculation frequency (0-1 ejaculation) during the six training days was registered and from the 63 male rats sexually trained from the first group and 32 from the second group, only 24 and 20 animals (respectively) that showed stable normal and low sexual performance and ejaculations respectively, were selected to run the pharmacological studies (see Supplementary Figures 1, 2). For the first group (selection for normal ejaculation rats), there was a significant week (time) effect F (7 . 154) = 13.86, p < 0.001, a significant week × genotype effect F (7 . 154) = 3.40, p < 0.01 and a significant genotype effect (F (1 , 22) = 23.81, p < 0.001). In SERT +/+ rats from week 3 onward they performed significant more ejaculations (all p-values are < 0.05) compared to the first 2 weeks (Figure 1 and Table 2). In SERT −/− rats only week 16-20 significantly differed (all p-values are < 0.05) from all other weeks (Figure 1 and Table 2). SERT −/− rats ejaculated significantly less compared to SERT +/+ rats in week 3 (p < 0.05), week 4 (p < 0.05), week 5 (p < 0.05), week 6 (p < 0.05), weeks 7-14 (0.05), and weeks 16-20 (p < 0.01).
For the second group of animals trained (for selection of slow ejaculating rats), there was a significant difference in weeks of training (F (10 , 180) = 3.453, p < 0.001). In week 11-14, SERT +/+ and SERT −/− rats had significant more ejaculations compared to all other weeks (all p-values < 0.01). No significant differences in time × genotype, and genotype effects were found during the training weeks (Figure 2 and Table 3).
We included in Figure 1 the saline data gathered in the pharmacological experiments performed on animals in group one (selection for normal ejaculating rats). The saline data obtained for all animals in weeks 7-15 (Exp. 1) were comparable to the last training data, but the saline data from the last (S15535) experiment (Exp. 2) (during week 16-20) showed significantly higher values. This "enhanced" baseline level of sexual behavior made us decide (because of possible ceiling effects) to repeat the S15535 experiment in rats with very low levels of sexual ejaculation activity (group two, Exp. 3: data shown in Table 3). In FIGURE 1 | Mean ejaculation frequencies (±SEM) over 6 weeks of training of male Wistar rats of group one (selection for normal ejaculating rats). Added are also the mean ± SEM of the saline data from experiment one (F13714 and F15599) and two (S15535) of group one. a: significantly different (p < 0.05) from week 1; b: significantly different (p < 0.05) from week 2;c: significantly different (p < 0.05) from week 3; d: significantly different (p < 0.05) from week 4; e: significantly different (p < 0.05) from week 5; f: significantly different (p < 0.05) from week 6; g: significantly different (p < 0.05) from week 7 to 15; *significantly different (p < 0.05) from SERT +/+ . Detailed statistical analyses are provided in Table 2.
In the slow ejaculating rats (Exp. 3), no significant differences were found in the majority of parameters measured, although a significant dose effect was found for the number of mounts  Table 2. a: significant difference (p < 0.05) compared to saline group, b: significant difference (p < 0.05) compared to 0.0025mg/kg group, c: significant difference (p < 0.05) compared to 0.01/mg/kg group. +Significant difference between SERT +/+ and SERT −/− (p < 0.05).

DISCUSSION
In the present study, after extensive training of the two genotypes studied (SERT +/+ and SERT −/− ), animals showed two different but stable sexual phenotypes, confirming earlier findings  where male SERT +/+ rats performed sexual behavior at a higher level than SERT −/− rats. Permanent changes in serotonergic processes in the central nervous system by removing the SERT protein from conception on Olivier et al., 2011), apparently leads to permanent changes in overt male sexual behavior in rats. The male rat sexual behavior paradigm used in the present studies has been developed over the last decades (Pattij et al., 2005;Chan et al., 2008;Olivier et al., 2011), specifically to test the effects of psychoactive drugs, including antidepressants (Waldinger and Olivier, 2005;Chan et al., 2010;Heijkoop et al., 2018). The paradigm is able to distinguish acute effects of drugs like the pro-sexual effects of 5-HT 1A receptor agonists (Pattij et al., 2005), but also the chronic inhibitory effects of SSRI antidepressants Bijlsma et al., 2014). Pro-sexual effects of drugs in male rat sexual behavior are reflected in the speed of onset of sexual activity toward a newly introduced female in behavioral estrus; reflected in a shorter interval to reach ejaculation (Andersson and Larsson, 1994), including reduced number of mounts and intromissions to reach ejaculation and enhanced number of ejaculations over a certain test period (in our case 30 min). Reduction of sexual behavior, e.g., by chronic antidepressants Bijlsma et al., 2014) has reversed effects. This chronic SSRI (antidepressant)induced profile of reduced male sexual behavior is comparable to the sexual behavior of SERT −/− rats and supports the hypothesis that male SERT −/− rats are modeling the sexual effects of chronic SSRI administration Olivier et al., 2011).
Several studies on SERT-genotypes in sexual behavior have been performed in at least three different labs (Utrecht, Groningen, Netherlands) and Hefei (China: Geng et al., 2019). In all three independent studies male SERT −/− rats display a significantly lower level of sexual behavior than SERT +/+ rats Geng et al., 2019;. It can be suggested that full absence of the SERT reduces the level of each individual rat's sexual behavior. It can be speculated that the resulting sexual phenotype of a SERT −/− rat may be derived from a certain basic sexual behavior that in some way is permanently inhibited when the SERT is absent from conception on. This can also be illustrated by the comparable ejaculation curves (# ejaculations after training) for both genotypes of which the SERT −/− rats are shifted to the left compared to the SERT +/+ rats . The typical distribution patterns of the # of ejaculations (or the 1st ejaculation latency times) in a large cohort of SERT-genotypes  supplies us with the possibility to behaviorally match animals with a certain genotype, e.g., only high versus low sexually performing animals, as we did previously in the study of tramadol effects on male sexual behavior (Esquivel-Franco et al., 2018).  Supplementary Table 4. a: significant difference (p < 0.05) compared to saline group, c: significant difference (p < 0.05) compared to 1/mg/kg group. +Significant difference between SERT +/+ and SERT −/− (p < 0.05).
In the present experiments we intentionally created (Exp. 2) two groups of SERT +/+ and SER −/− rats with low (not statistically different) levels of sexual behavior in order to circumvent possible interference of high versus low rates of behavior.
Two biased 5-HT 1A receptor agonists, the preferential 5-HT 1A auto-receptor agonist F13714 (Assié et al., 2006; Becker et al., 2016) and the preferential 5-HT 1A heteroreceptor agonist F15599 (Newman-Tancredi et al., 2009;Becker et al., 2016) were tested in SERT +/+ and SERT −/− rats. Both compounds induced pro-sexual activity in SERT +/+ and SERT −/− rats (for overview see Table 4). F13714 is considerably more potent than F15599 in eliciting the pro-sexual effects, but the similarity of the response of both compounds on male sexual behavior suggests that both compounds share comparable mechanisms of action in evoking sexual behavior. This may point to an autoreceptor-mediated effect. Unfortunately, full dose-response curves of this pro-sexual effect were not available for both compounds making definite conclusions impossible. In F13714-treated SERT −/− rats the dose-response curve of prosexual activity was shifted to the right compared to SERT +/+ rats, but this was not the case in F15599 treated rats where the sexual inhibiting doses were comparable in both genotypes. 5-HT 1A receptor stimulation by "non-selective" (with regard to pre-and post-synaptic receptors) 5-HT 1A receptor agonists like 8-OH-DPAT, flesinoxan, buspirone, ipsapirone, and others (Olivier et al., 1999) have pro-sexual effects in wildtype rats (Snoeren et al., 2014 for review), but no studies were performed before where the specific contributions of 5-HT 1A autoreceptors or 5-HT 1A heteroreceptors (or both) are investigated. S15535, an auto-receptor selective 5-HT 1A receptor agonist and heteroreceptor-selective 5-HT 1A receptor antagonist, did not have any effects on male sexual behavior of SERT +/+ and SERT −/− rats, neither in normal ejaculating (on average 1-2 ejaculations/30 min; group 1) nor in slow ejaculating (0-1 ejaculations/30 min; group 2) rats. We conclude that S15535 behaves as a "silent" 5-HT 1A receptor ligand in male rat sexual behavior. The prototypal 5-HT 1A receptor agonist (±) or (+)-8-OH-DPAT, a non-selective auto-receptor and heteroreceptor agonist (Larsson et al., 1990), has strong and dose-dependent pro-sexual effects (Mos et al., 1991;Chan et al., 2011;Snoeren et al., 2014). This pro-sexual effect can be fully antagonized by the 5-HT 1A receptor antagonist WAY100,635, a behaviorally silent compound (de Jong and Neumann, 2015). In male SERT −/− rats  8-OH-DPAT had pro-sexual effects, although (like the biased agonist F13714 in the present study) the doseresponse curve was shifted to the right compared to SERT +/+ rats. The lack of any behavioral effect of S15535 in either SERT +/+ or SERT −/− rats is rather puzzling. Apparently, 5-HT 1A receptor antagonistic activity on 5-HT 1A heteroreceptors in SERT −/− rats did not cause inhibition of male sexual behavior like WAY100,635 treatment . The stimulating effect of F13714 and F15599 in male sexual behavior in both SERT +/+ and SERT −/− rats is also quite puzzling, because it makes explanations in term of pre-or post-synaptic 5-HT 1A receptor mechanisms involved, troublesome. However, it remains possible that the preferential post-synaptic 5-HT 1A receptor agonist F15599, at higher doses (like in this experiment) also displays some presynaptic autoreceptor agonistic activity. In that case F15599 does not appear the specific tool to selectively activate post-synaptic 5-HT 1A heteroreceptors.
How do the sexual data obtained with these three serotonergic ligands compare to their effects in other behavioral systems? The research group of De Boer (de Boer and Newman-Tancredi, 2016) has tested these (and other) ligands extensively in male rat models of offensive aggression in Wildtype Groningen (WTG) rats. In male rat offensive aggression (de Boer et al., 1999(de Boer et al., , 2000 8-OH-DPAT potently and dose-dependently reduced offensive aggression but also induces strong sedative-like behaviors. Because 5-HT 1A receptor agonists induce a so-called serotonin-5-HT 1A syndrome, characterized by Lower Lip Retraction (LLR), Forepaw Treading (FPT), and Flat Body Posture (FBP), it is not completely clear whether this sedative-like activity is similar to these serotonergic behaviors. These anti-aggressive and other effects of 8-OH-DPAT can be fully antagonized by WAY100,635 (de Boer et al., 1999(de Boer et al., , 2000, a silent antagonist in offensive aggression. F13714, F15599, and S15535 all reduce offensive aggression (de Boer and Newman-Tancredi, 2016). Both F13714 and F15599 induce a serotonergic-5-HT 1A syndrome in rats (Newman-Tancredi et al., 2009;Assié et al., 2010;Jastrzȩbska-Wiȩsek et al., 2018). S15535 does not induce the serotonergic-5-HT 1A syndrome at all (de Boer and Newman-Tancredi, 2016;Jastrzȩbska-Wiȩsek et al., 2018) and also has no sedativelike activity in offensive aggression (de Boer et al., 2000). WAY100,635 antagonized the anti-aggressive action of S15535, F15599, and F13714 (de Boer and Newman-Tancredi, 2016).
If the mechanisms of action of the three 5-HT 1A ligands as extensively investigated by various research groups are true, mechanistic interpretations of the behavioral effects found in male sexual behavior are rather difficult to make. Serotonergic 5-HT 1A auto-receptors in the raphe nuclei are generally considered as, upon activation, leading to inhibition of cell firing and consequently a decrease of serotonin release. Subsequently, all post-synaptic 5-HT (hetero) receptors (including 5-HT 1A heteroreceptors) receive diminished or no stimulation by serotonin and depending on the coupling of the post-synaptic receptor to different transduction mechanisms the neuron involved will be activated or inhibited. Serotonin is also known to crosstalk with non-serotonergic systems which may exert effects on (sexual) behavior as well (e.g., Blier, 2001). In case of a non-selective 5-HT 1A receptor agonist like 8-OH-DPAT, next to its inhibiting action on the serotonergic neuron, direct 5-HT 1A heteroreceptor stimulation still occurs leading to post-synaptically mediated effects, like the serotonergic-5-HT 1A behavioral syndrome (Berendsen et al., 1990;Jastrzȩbska-Wiȩsek et al., 2018). In the case of F13714, a relatively selective (compared to heteroreceptor) 5-HT 1A auto-receptor agonist (Assié et al., 2006) potently facilitated sexual activity in male SERT +/+ rats suggesting that pro-sexual activity is related to activation of 5-HT 1A auto-receptors. The relatively selective 5-HT 1A heteroreceptor agonist F15599 also facilitated male sexual activity in SERT +/+ rats. The difference in potency (factor 256 difference) to obtain the pro-sexual activity (at the lowest effective dose) can possibly be explained by the difference of the in vitro and in vivo affinity and efficacy of both compounds on 5-HT 1A receptors (Assié et al., 2010;Newman-Tancredi, 2011;Jastrzȩbska-Wiȩsek et al., 2018). This might be taken as suggestive that both compounds exert pro-sexual activity via activation of 5-HT 1A auto-receptors. Strangely enough, both compounds also activate the serotonergic-5-HT 1A syndrome (Newman-Tancredi, 2011;Becker et al., 2016). The 5-HT 1A autoreceptor agonist S15535 does not induce pro-sexual behavior, neither in normal nor in sexually slow ejaculating rats. Whether blocking of post-synaptic 5-HT 1A heteroreceptors antagonizes the expected pro-sexual effect of the auto-receptor stimulation is rather difficult to envisage. This would assume a rather high level of basal activity of 5-HT 1A heteroreceptors involved in sexual behavior. Interestingly, Pattij et al. (2005) showed that slow, normal and rapid ejaculating rats showed increased ejaculations after treatment with 8-OH-DPAT; however, when rats were re-tested 1 week after this 5-HT 1A receptor agonist administration all phenotypes returned to ejaculatory behavior levels found before the 8-OH-DPAT treatments. In the present study we found that during the weeks where treatment with S155355 were administered, the saline groups (and thus baseline levels) showed significant higher ejaculation frequencies compared to the ejaculation frequencies during the training weeks. This might suggest that pro-sexual effects due to 5-HT 1A receptor agonist can be long-lasting, most likely due to alterations in the 5-HT 1A receptors. Further research is warranted to investigate how long this effect would persist and whether it is, 1 week after all treatments with 5-HT 1A receptor agonists, and without saline treatment, still present.
SERT −/− rats, a model of permanently changed serotonergic activity in the brain (Homberg et al., 2007) and associated with an altered sexual phenotype  may be helpful in explaining the behavioral effects found for the three compounds. Chan et al. (2011) have found that 8-OH-DPAT has pro-sexual effects in male SERT −/− rats, although the dose-response curve has been shifted to the right compared to SERT +/+ rats. Remarkably, WAY100,635, a non-selective 5-HT 1A receptor antagonist and without any behavioral effects in SERT +/+ males, was (dose-dependently) inhibitory in SERT −/− rats. WAY100,635 was able to completely antagonize the prosexual effects of 8-OHDPAT in SERT +/+ rats but only partially in SERT −/− rats . We concluded from these data that complete absence of SERT molecules had led to alterations in 5-HT 1A receptor functioning, hypothesizing that one pool of 5-HT 1A receptors mediates pro-sexual effects of 5-HT 1A receptor stimulation and is not (de)sensitized, whereas another pool of 5-HT 1A receptors, mediating the inhibitory effects of antagonized 5-HT 1A receptors seems sensitized in the SERT −/− rats. The hypothesis of two differentially regulated 5-HT 1A receptor pools in SERT −/− rats has also been found in autonomic regulation of body temperature and stress . The findings with F15599 and F13714 in the SERT −/− rats cannot be explained in terms of action on different 5-HT 1A receptor pools. If any, both compounds seem to activate the pool mediating the pro-sexual effects. The 5-HT 1A heteroreceptor antagonistic effects of S15535 do not lead to inhibition of male sexual behavior in the better performing (normal ejaculating) SERT −/− rats, as was the case for WAY100,635 in the Chan et al. (2011) study.
Our expectation that biased 5-HT 1A receptor agonists and a mixed 5-HT 1A presynaptic receptor agonist and post-synaptic antagonist might help to reveal the potential contribution of these different 5-HT 1A receptors was too optimistic. The mechanisms of action of the respective molecules are probably to complex, especially in vivo in complicated networks, where 5-HT 1A receptors interact with various other neurotransmitter systems in the modulation of male sexual behavior.

CONCLUSION
The data collected with the pharmacological experiments show that selective (preferential) pre-and postsynaptic 5-HT 1A receptor agonists possess pro-sexual effects in SERT +/+ and SERT −/− , although the response is diminished in SERT −/− animals, most likely due to desensitization of 5-HT 1A receptors. The pharmacological experiment with S15535 compared with previous experiments performed in aggression lacked any sexual behavioral effect. Further experiments are needed to explore whether separate neurobiological substrates at the 5-HT 1A receptors level exist.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
This study was carried out in accordance with the principles of the EU Directive 2010/63/EU.

AUTHOR CONTRIBUTIONS
DE-F, BO, and JO contributed with conception and design of the work. DE-F carried out all the experimental work, data collection and analysis, and draft work. DE-F, BO, SB, and JO contributed to the interpretation of the data and results, made sure all parts of the work were appropriately investigated and resolved. DE-F, BO, SB, MW, and JO contributed on revising critically the intellectual content, accountability and accuracy of the work, and gave approval for the publication of the content.