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- Materials and methods
Studies in mice with targeted deletions of tachykinin genes suggest that tachykinins and their receptors influence emotional behaviors such as aggression, depression and anxiety. Here, we investigated whether TAC1- and TAC4-encoded peptides (substance P and hemokinin-1, respectively) and the neurokinin-1 receptor (NK-1R) are involved in the modulation of sexual behaviors. Male mice deficient for the NK-1R (TACR1 −/−) exhibited decreased exploration of female urine in contrast to C57BL/6 control mice and mice deficient for NK-1R ligands such as TAC1 −/−, TAC4 −/− and the newly generated TAC1 −/− /TAC4 −/− mice. In comparison to C57BL/6 mice, mounting frequency and duration were decreased in male TACR1 −/− mice, while mounting latency was increased. Decreased preference for sexual pheromones was also seen in female TACR1 −/− mice. Furthermore, administration of the NK-1R-antagonist L-703,606 decreased investigation of female urine by male C57BL/6 mice, suggesting an involvement of NK-1R in urine sniffing behavior. Our results provide evidence for the NK-1R in facilitating sexual approach behavior, as male TACR1 −/− mice exhibited blunted approach behavior toward females following the initial interaction compared with C57BL/6 mice. NK-1R signaling may therefore play an important role in pheromone-induced sexual behavior.
Tachykinins are a family of neuropeptides that share the C-terminal motif FXGLM-NH2. In mouse, TAC1 encodes substance P (SP) and neurokinin A (NKA) through alternative splicing. TAC2 produces neurokinin B (NKB), and TAC4 encodes hemokinin-1 (HK-1). Tachykinins mediate their actions through three G-protein-coupled receptors, neurokinin-1 receptor (NK-1R, TACR1), NK-2R (TACR2) and NK-3R (TACR3). SP and HK-1 are the preferred, endogenous ligands for NK-1R. NKA preferentially binds to NK-2R and NKB to NK-3R. However, each ligand can interact with all receptors with varying affinity (Patacchini & Maggi 2001).
SP and NK-1Rs are expressed in various brain regions involved in social and sexual behaviors, including the olfactory bulb, striatum, amygdala and hypothalamic nuclei (Allen Brain Atlas, www.brain-map.org). Owing to the involvement of tachykinins in the regulation of emotional behaviors such as the reduced aggression of TACR1−/− mice in male–male interaction studies (De Felipe et al. 1998), we decided to investigate male–female interaction and social/sexual behavior in tachykinin null-mutant mice, including TACR1−/−, TAC1−/−, TAC4−/− and the newly generated TAC1−/−/TAC4−/−‘double-knockout mice’, which are deficient for TAC1- and TAC4-encoded peptides. Mice deficient for TACR1, but not TAC1 and/or TAC4, exhibited decreased interest in sexual pheromones. Furthermore, male TACR1−/− mice displayed diminished approach, courtship and sexual behaviors, suggesting a role for NK-1R signaling in these complex behaviors.
- Top of page
- Materials and methods
In this study, we report that the NK-1R plays a role in the modulation of sexual behavior. Male NK-1R-deficient mice (TACR1−/−), but not mice deficient for tachykinin peptides, exhibited a significant decrease in olfactory investigation of female urine compared with C57BL/6 control mice. As TACR1−/− mice differentiated between odors, and because olfaction plays an essential role in rodent behavior, the decreased sniffing of female urine may indicate social and/or sexual behavioral deficits. For most animals, successful recognition of chemosensory odor cues is critical for social communication as well as for social and sexual behavior. Urine and sexual attractants such as pheromones are important inducers of these behaviors (Wang et al. 2008).
During courtship, male mice emit complex song-like USVs, which are interpreted as a means of social communication. They are frequently observed during sexual behavior and are thought to attract the female to the male. USVs can be induced by urine and pheromones (Wang et al. 2008). As the decreased exploration of female urine exhibited by male TACR1−/− mice may be interpreted as decreased interest in female urine and/or pheromones, we investigated whether the quantity of USVs during male–female interaction was altered. Although not statistically significant, TACR1−/− males emitted fewer USV calls than C57BL/6 males. Consistent with the decreased investigation of female urine, TACR1−/− males further displayed less vigorous mating behavior, as determined by decreased mounting frequency and duration and increased mounting latency compared with C57BL/6 males. The blunted approach behavior of TACR1−/− males toward females following the initial interaction suggests a role for NK-1R in facilitating sexual approach behavior and in pheromone-induced sexual behavior.
The idea that dopamine is involved in the processing of information derived from rewarding stimuli is commonly accepted (Martinez-Hernandez et al. 2012). However, the precise role of dopamine in the reward system is widely debated (Berridge & Robinson 1998, 2003; Nicola 2010). As urine pheromones can represent a rewarding cue for mice, the decreased sniffing of female urine could be interpreted as a deficiency to engage in reward-seeking behavior. In fact, the FUST has recently been described as a test to assess changes in reward-seeking behavior (Malkesman et al. 2010). TACR1−/− mice are known to display deficiencies in reward-seeking behavior. For example, TACR1−/− mice failed to acquire a response to self-administered morphine, suggesting a role for NK-1R in mediating the rewarding properties of opiates (Ripley et al. 2002). TACR1−/− mice were further reported to consume lower amounts of alcohol compared with controls (George et al. 2008; Thorsell et al. 2010). The failure to develop a preference for an environment paired with morphine or alcohol suggests that the rewarding effects of these substances are impaired in TACR1−/− mice. The results presented here could therefore be interpreted as the failure to develop a preference for an environment paired with sexual pheromones. As sexual behaviors are driven by pheromone cues, this impairment could consequently be responsible for the blunted sexual behavior of male NK-1R-deficient mice.
Impairment to engage in reward-seeking behavior points to abnormalities of brain networks that mediate reward signals. Neurotransmitters such as dopamine and the endogenous opioids endorphin, enkephalin and dynorphin mediate reward and reward-induced behaviors through opioid receptors. Pheromone-induced reward is mediated by opioidergic neurotransmitters as opioidergic inhibitors suppressed the processing of pheromone signals leading to inhibition of sexual behavior (Agustin-Pavon et al. 2008). The µ-opioid receptor (MOR) is the primary target mediating the rewarding effects of morphine and alcohol, which have been reported to be disrupted in TACR1−/− mice (Baek et al. 2010; Ripley et al. 2002; Thorsell et al. 2010). The NK-1R has been shown to regulate MOR signaling in vitro, providing a link between NK-1R and reward signals (Yu et al. 2009).
Behavioral studies have shown greater locomotor activity in TACR1−/− mice compared with controls and while d-amphetamine increased locomotor activity of wild-type mice, it reduced that of TACR1−/− mice. Furthermore, while basal dopamine efflux in the dorsal striatum was similar in wild-type and TACR1−/− mice, administration of d-amphetamine resulted in an increase in dopamine efflux in wild-type but not TACR1−/− mice (Yan et al. 2010). This biochemical imbalance in combination with behavioral changes such as hyperactivity and the disrupted response to rewarding stimuli resembles the human neurological condition ‘attention deficit hyperactivity disorder’ (ADHD). Symptoms of ADHD are hyperactivity, impulsivity, distractibility and inattention. Genetic studies showed polymorphisms in the human TACR1 gene in ADHD patients, suggesting that individuals with TACR1 mutations may be susceptible to ADHD (Yan et al. 2009). We were concerned that two odors being present at the same time in the FUST may be the reason for the decreased sniffing of female urine exhibited by TACR1−/− mice, simply as a result of ADHD-like behavior. We therefore included female urine in the habituation/dishabituation test, which confronts the animal with only one odor at a time. There was no difference in the outcome of the test. Although TACR1−/− mice displayed significant habituation/dishabituation, which suggests that they can differentiate between odors, they showed significantly decreased sniffing of female urine compared with C57BL/6, confirming that TACR1−/− mice show a specific disinterest in female urine.
Whether the decreased exploration of female urine, the fewer USV calls and the decreased mounting behavior of TACR1−/− males presented here are due to impaired pheromone processing, a deficiency in the rewarding effects of pheromones and thus diminished pheromone reward-seeking behavior or other behavioral abnormalities remains to be investigated. It is important to note that testosterone levels of male TACR1−/− mice did not differ from C57BL/6 mice. Furthermore, TACR1−/− mice are fertile and produce offspring with litter sizes comparable to that of other strains.
On the basis of the biochemical imbalance in TACR1−/− mice involving dopamine and d-amphetamine, we reasoned that d-amphetamine treatment may affect male sexual behavior differently in C57BL/6 controls and TACR1−/− mice. We show that d-amphetamine treatment decreased female urine sniffing in male C57BL/6 but not TACR1−/− mice. Consistent with our data obtained from male C57BL/6 mice, d-amphetamine treatment abolished the preference of female mice for male pheromones, suggesting that dopamine has an inhibitory role on pheromone processing (Lanuza et al. 2008). As TACR1−/− mice lack the increase in striatal dopamine efflux that occurs post-d-amphetamine administration (Yan et al. 2010), which in wild-type mice inhibits pheromone processing (Lanuza et al. 2008), this would offer an explanation as to why d-amphetamine decreased pheromone preference in male C57BL/6 but not TACR1−/− mice. However, because the urine sniffing times are very low in TACR1−/− mice, the absence of a drug effect being the result of a floor effect cannot be excluded.
Surprisingly, neither male TAC1−/−/TAC4−/−‘double-knockout mice’ lacking SP, NKA and HK-1 nor the single-knockout mice exhibited the same behavioral deficit as male TACR1−/− mice. In agreement with our findings, male SP-deficient mice responded to female urine in a C57BL/6-like fashion (Bilkei-Gorzo et al. 2002). All mutant mice used in our study were on the same genetic background (C57BL/6), thereby excluding variations due to vastly different background genetics, which has been shown to affect phenotypic changes (McCutcheon et al. 2008). Our findings are intriguing as SP and HK-1 are the major ligands for NK-1R. As NK-1R-mediated responses are mainly thought of as being SP mediated, our findings raise the possibility of other ligands acting through NK-1R when SP is not available. As TAC1−/−/TAC4−/− mice are deficient for SP, NKA and HK-1, the only tachykinin peptide left for consideration is NKB. Compensatory mechanisms involving NKB and functional redundancy may explain the lack of phenotypic alterations in TAC1−/−/TAC4−/− mice.
To confirm that the impaired olfactory investigation of female urine is due to NK-1R involvement rather than a ‘flanking allele problem’ and to verify that the use of C57BL/6 as controls instead of wild-type litter mates is a valid experimental approach, we performed the FUST on male C57BL/6 mice treated with the NK-1R-antagonist L-703,606. Treatment significantly decreased the female urine sniffing behavior of male C57BL/6 mice, indicating that the observed behavioral differences are not due to maternal effects that may have occurred because the parents of the control and mutant subjects had different genotypes, but rather are due to the absence of NK-1R, specifically. However, NK-1R antagonist treatment could not fully mimic the deficits seen in TACR1−/− mice. Given that neither TAC1−/− nor TAC1−/−/TAC4−/− mice showed a TACR1−/−-like phenotype, this finding is not surprising as NK-1R-antagonists were designed to block SP signaling, which does not seem to be solely responsible for the NK-1R-mediated deficiencies described here. A similar conclusion was made when TAC1−/− and TACR1−/− mice were compared in a migraine model. Whereas TACR1−/− mice were protected from dura-mater vascular permeability, a symptom of migraine headaches, TAC1−/− mice had intact vascular permeability, suggesting the involvement of a ligand other than SP/NKA (Kandere-Grzybowska et al. 2003). Low efficiency of NK-1R-antagonists in rodents has been attributed to low potency as most antagonists have been developed for human use. Owing to the mounting evidence of NK-1R involvement in a range of diseases and disorders, a reassessment of NK-1R-antagonist pharmacology may be warranted.
In conclusion, we show that disruption of the TACR1 gene leads to blunted sexual approach behavior in male mice following the initial encounter of a female. Our data therefore provide evidence for an important role of NK-1R signaling in the regulation of pheromone-induced sexual behavior.