Balanced release of neuropeptide FF and gonadotropin-releasing hormone 3 modulates male sexual behavior

Animals properly perform sexual behaviors by using multiple sensory cues. However, neural mechanisms integrating multiple sensory cues and regulating motivation for sexual behaviors remain unclear. Here, we focused on peptidergic neurons, terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons, which receive inputs from various sensory systems and co-express neuropeptide FF (NPFF) in addition to GnRH. Our behavioral analyses using knockout medaka of GnRH (gnrh3) and/or NPFF (npff) demonstrated that some sexual behavioral repertories were ‘delayed’, not disrupted, in gnrh3-/- and npff-/- males, while the double knockout showed normal behaviors. We also found anatomical evidence to show that both neuropeptides modulate the sexual behavior-controlling brain areas. Furthermore, we demonstrated that NPFF activates neurons in the preoptic area via indirect pathway, which is considered to induce the increase in the motivation for male sexual behaviors. Considering these results, we propose a novel mechanism by which balanced release of co-existing peptides is important for the neuromodulatory function of TN-GnRH neurons in the control of behavioral motivation. Our results may go a long way toward understanding the functional significance of peptidergic neuromodulation in response to external environments.


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Animals properly perform sexual behaviors by using multiple sensory cues. However, neural 25 mechanisms integrating multiple sensory cues and regulating motivation for sexual behaviors 26 remain unclear. Here, we focused on peptidergic neurons, terminal nerve gonadotropin-releasing 27 hormone (TN-GnRH) neurons, which receive inputs from various sensory systems and co-express 28 neuropeptide FF (NPFF) in addition to GnRH. Our behavioral analyses using knockout medaka 29 of GnRH (gnrh3) and/or NPFF (npff) demonstrated that some sexual behavioral repertories were 30 'delayed', not disrupted, in gnrh3 -/and npff -/males, while the double knockout showed normal 31 behaviors. We also found anatomical evidence to show that both neuropeptides modulate the 32 sexual behavior-controlling brain areas. Furthermore, we demonstrated that NPFF activates 33 neurons in the preoptic area via indirect pathway, which is considered to induce the increase in   Yamamoto et al., 1997). In addition, this neuron is anatomically suggested to receive 52 sensory information (Yamamoto and Ito, 2000).

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Here, we performed behavioral analyses using gnrh3 knockout (gnrh3 -/-) and/or npff knockout 78 (npff -/-) medaka and found that NPFF and GnRH3 modulate male sexual behaviors. In addition, 79 to localize the target neurons of NPFF or GnRH3 in the brain, we anatomically analyzed the supplement 2B) among all the pairs, it is suggested that gnrh3 -/male are not able to show sexual ends (RACE), we found three genes as NPFF receptors and identified gpr147 (so-called NPFFR1, 139 Bonini et al., 2000), gpr74-1 and gpr74-2 (so-called NPFFR2, Bonini et al., 2000) (Figure 4-140 figure supplement 1). All of these receptors were shown to function as G-protein coupled 141 receptors, whose ligand is NPFF (Figure 4-figure supplement 2). Then, by in situ hybridization 142 of these genes, we found that these receptors are expressed broadly in the brain ( Figure 4A, 143 abbreviation of brain region in Supplementary file 1). Because TN-GnRH neurons do not project 144 to the pituitary, we analyzed brain regions except the pituitary. In Vv and Vs, which are the brain 145 region involved in the control of sexual behavior (Koyama et al., 1984, Satou et al., 1984, gpr74-146 2 and gpr147 were abundantly expressed, respectively ( Figure 4B and 4C, abbreviation of brain 147 nuclei is described in Supplementary file 1). In addition, in POA and hypothalamus, all subtypes 148 of NPFF receptors were expressed ( Figure 4D). gpr74-1 was expressed in the dorsal and ventral early genes, egr1, in npff -/male with that in WT male. egr1 expression is suggested to represent 176 neuronal activities during 30 min before sacrifice (Burmeister and Fernald, 2005). We made pairs 177 according to the behavioral test of npff -/described above, sacrificed the male, and dissected out 178 the brains 25 -28 min after removing the transparent separation, when the motivation for sexual 179 behavior in WT male was observed to be high in the present behavioral analysis (see Figure 2).

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Because WT showed more egr1-expessing neurons only in the ventral part of preoptic area 181 (vPOA) than npff -/male among other brain regions, we examined co-expression of egr1 and 182 gpr74-1, which is the npff receptor gene expressed in the vPOA ( Figure 4D). As shown in Figure   183 6A and 6B, the percentage of egr1-expessing neurons in the vPOA of npff -/male was less than 184 that of WT, while the difference between the percentage of gpr74-1-expessing neurons in npff -/-185 and that in WT was not significant ( Figure 6C). On the other hand, we found a small number of 186 egr1 and gpr74-1 co-expressing neurons in both genotypes (WT: 30 ± 8 cells, npff -/-: 13 ± 7 cells), 187 although the difference in the percentage of egr1and gpr74-1-coexpessing neurons was not 188 significant between the two ( Figure 6D). It should be noted that all WT males spawned within 6 189 min after removing the transparent separation, while five out of six npff -/males failed to spawn

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The expression level of npff in gnrh3 -/medaka is lower than that in WT medaka

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Our behavioral analyses suggested that NPFF and GnRH3 may facilitate the motivation for the 196 control of male sexual behaviors in slightly different manners. Therefore, to examine the 197 relationship of GnRH3 and NPFF, we analyzed the expression of npff or gnrh3 in npff -/-, gnrh3 198 -/-, and WT medaka. As shown in Figure 7A, the expression level of npff in npff -/was lower than 199 that in WT, and that of gnrh3 in gnrh3 -/was lower than that in WT, which suggests that nonsense-

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In the present study, we also showed that GnRH receptors are expressed mostly in the sensory 303 processing areas ( Figure 5A). Two subtypes of GnRH receptors, gnrhr1 and gnrhr3, were 304 expressed in the Dp, which is the olfactory and gustatory processing areas (Murakami et al., 1983, 305 Yoshimoto et al., 1998). In addition, gnrhr1-expressing neurons were localized in the NI, which

Data availability
The sequences of NPFF receptors were deposited in GenBank under the accession number 572 AB697148 for GPR147, AB697149 for GPR74-1, and AB697150 for GPR74-2. All data are 573 included in the manuscript and supporting source data files. Source data files have been 574 provided for Figure 2, Figure 3, Figure4-figure supplement2, Figure5-figure  575 supplement1, Figure 6, and Figure 7. 576 577 Tables 578 Table 1