Chie Matani, Assistant Professor, Department of Biological Sciences

Yoshitaka Oka, Professor Emeritus Professor, Department of Biological Sciences

Key points of the presentation

• We have revealed that two neuropeptides ^{(Note 2)} GnRH3 and NPFF ^{(Note 3} ) made by the same neurons ^{(Note 1)} in the brain both regulate motivation in male sexual behavior.
• In addition to discovering the function of NPFF in behavior, which had been unknown, this study clarified the mechanism in the brain by which sexual behavior is successfully regulated when both neuropeptides act in the brain in a balanced manner.
• The clarification of how multiple neuropeptide-producing neurons regulate behavioral motivation is expected to advance our understanding of the mechanisms in the brain that control animal behavior.

Summary of Presentation

In general, male and female animals recognize each other as the opposite sex using various information from inside and outside the body, and reproduce by engaging in sexual behavior during the breeding season to leave offspring. However, much has remained unknown about the mechanisms in the brain from the time the brain receives various information until the animal exhibits sexual behavior.

A research group at The University of Tokyo's Graduate School of Science focused on the terminal GnRH neurons, which have been reported to receive various sensory inputs, and manipulated the genes for GnRH3 and NPFF, two neuropeptides produced by these neurons, to create a killifish with impaired function and analyzed its sexual behavior in detail. The results showed that only one of the peptides was dysfunctional. Although the male killifish in which only one of the peptides had been lost eventually became sexually active, much of the multi-part sexual behavior occurred later than in the wild type. On the other hand, male killifish with loss-of-function of both peptides did not exhibit the noticeable delay in sexual behavior that was observed in the single-peptide loss-of-function killifish.

These results suggest that multiple neuropeptides made by the same neuron act in the brain in a balanced manner to properly regulate male sexual behavior. The results of this research are expected to lead to a better understanding of the mechanisms in the brain that flexibly regulate the motivation of animal behavior according to the situation.

Content of Presentation

In general, males and females recognize each other as the opposite sex using various sensory information such as olfactory, visual, auditory, and somatosensory information, and they use hormonal information in their bodies to perform courtship behavior at the appropriate timing during the breeding season in order to leave offspring. However, there are still many unknowns regarding the mechanism in the brain by which the brain accepts and integrates various sensory information and then displays sexual behavior. Behavior is mainly regulated in the brain, and peptide neurons ^{(Note 2)} are thought to play a key role in the process of integrating information and displaying appropriate behavior.

In this study, we focused on peptide neurons called terminal nerve (TN-) gonadotropin-releasing hormone (GnRH) neurons. These neurons are evolutionarily conserved in almost all vertebrates and have been suggested to receive neural input from multiple brain regions that process sensory information. They also extend their neuronal projections over a wide area of the brain, including brain regions involved in sexual behavior. Furthermore, this neuron has been reported to produce and release not only GnRH3, from which it derives its name, but also a neuropeptide called NPFF ^{(Note 3} ). On the other hand, despite the fact that these neuropeptides have been discovered more than 50 years ago, it was not well understood whether they are involved in sexual behavior. Therefore, using medaka fish that show a typical sexual behavior pattern, we genetically engineered two neuropeptides produced by these neurons, GnRH3 and NPFF, to produce medaka fish in which the peptide function was lost, and analyzed their sexual behavior in detail and quantitatively. Specifically, one male and one female killifish were separated the day before using a transparent partition, and their sexual behavior was analyzed for 25 minutes after the partition was removed the next morning.

The results showed that male medaka that had lost function of only GnRH3 or NPFF were eventually able to perform sexual behavior, but much of their multi-part sexual behavior occurred later than in the wild type (Figure 1). On the other hand, male killifish with loss of both GnRH3 and NPFF function showed less noticeable delays in sexual behavior as observed in killifish with loss of single function (Figure 1).

Figure 1: Time until egg release occurs in medaka individuals with loss of function of the neuropeptide expressed in TN-GnRH neurons.
a. Egg release behavior of medaka. When female and male killifish are placed in the same tank, they exhibit stereotyped sexual behavior and fertilization occurs when the female eventually releases eggs and the male releases sperm. b. Pairs of killifish in which the neuropeptide NPFF was loss of function and wild-type heterosexual pairs showed slower egg release times. c. Loss of neuropeptide GnRH3 caused a delay in egg release time in male killifish and wild-type female pairs, whereas loss of both NPFF and GnRH3 caused a delay in egg release time in killifish and wild-type opposite-sex pairs. The delay in egg release time was not noticeable in pairs of killifish with loss of both NPFF and GnRH3 function and wild-type heterozygotes. (* and *** indicate statistically significant delays when compared to wild-type pairs.)

Furthermore, analysis of the brain regions where these neuropeptide-binding receptors are expressed revealed that they are expressed in a wide range of brain regions, including the brain regions Vv, Vs, and POA, which are known to regulate sexual behavior. In addition, the researchers analyzed the brain regions where early response genes, which are indicators of neural activity, are expressed by pairing untreated female and wild-type male killifish, and by pairing the untreated male and wild-type killifish, both of which were observed to have delayed behavior. The results showed that the expression of early response genes in the brain region called POA was lower in the NPFF-less male medaka than in the wild-type medaka.

These results indicate that without the neuropeptide GnRH3 or NPFF expressed in TN-GnRH neurons, the animals are ultimately capable of sexual behavior but the onset of behavior is delayed, suggesting that the loss of these peptides produces "reduced motivation for sexual behavior". In medaka in which both peptides were lost, the delay in behavior observed in medaka in which only one of the peptides was lost was less noticeable. These results suggest that GnRH3 and NPFF, each of which is released from TN-GnRH neurons, may act in a balanced manner on brain regions involved in sexual behavior, thereby appropriately regulating male sexual motivation (Figure 2). Furthermore, this study is the first to show that the NPFF, whose role in behavior in particular has been unclear, functions in regulating motivation for sexual behavior in animals. Thus, it was found that motivation for animal behavior is regulated by multiple neuropeptides produced by the same neuron.

Figure 2: Working hypothesis of the mechanism regulating motivation for male sexual behavior via GnRH and NPFF.
We found that GnRH3 and NPFF receptors are expressed in brain regions that process sensory information and those that regulate sexual behavior. Therefore, it is conceivable that GnRH3 and NPFF each increase motivation for sexual behavior in males by modulating the brain regions that are involved in integrating sensory information and ultimately controlling sexual behavior in a balanced manner. (Solid lines indicate strong effects; dotted lines indicate weak effects.)

The results of this research are expected to lead to a better understanding of the general mechanism in the brain that flexibly regulates the motivation of animal behavior according to the situation.

This research was supported by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (17K15157, 20H03071, 24570067, 18H04881, 18K19323, 26221104).

Journals

Journal name	Endocrinology
Title of paper	Co-existing neuropeptide FF and gonadotropin-releasing hormone 3 coordinately modulate male sexual behavior
Author(s)	Chie Umatani*, Nagisa Yoshida, Eri Yamamoto, Yasuhisa Akazome, Yasutaka Mori, Shinji Kanda, Kataaki Okubo, Yoshitaka Oka*
DOI Number	10.1210/endocr/bqab261
Abstract URL	https://academic.oup.com/endo/article/163/2/bqab261/6486464

Terminology

Note 1 Neuron .

Synonymous with neuron. It has many neurites, from which neuropeptides and other chemical substances are released, and signals are exchanged between neurons to produce brain functions such as sensation and movement. ↑up

Note 2 Neuropeptides and peptide neurons

Peptides are molecules composed of multiple amino acids that act as hormones and bioactive substances in the brain. Peptides produced and released by neurons are called neuropeptides. Peptide neurons are neurons that produce and release them. ↑up

Note 3 GnRH3 and NPFF

Abbreviations of neuropeptides called gonadotropin-releasing hormone 3 and neuropeptide FF, respectively. Both neuropeptides are evolutionarily conserved in vertebrates. In medaka TN-GnRH neurons, GnRH3 is produced, one of the three types of GnRH known to exist in vertebrates (GnRH1-3). ↑up