Mating Behavioral Function of Preoptic Galanin Neurons Is Shared between Fish with Alternative Male Reproductive Tactics and Tetrapods

Abstract

Understanding the contribution of neuropeptide-containing neurons to variation in social behavior remains critically important. Galanin has gained increased attention because of the demonstration that galanin neurons in the preoptic area (POA) promote mating and parental care in mammals. How widespread these mechanisms are among vertebrates essentially remains unexplored, especially among teleost fishes, which comprise nearly one-half of living vertebrate species. Teleosts with alternative reproductive tactics exhibit stereotyped patterns of social behavior that diverge widely between individuals within a sex. This includes midshipman that have two male morphs. Type I males mate using either acoustic courtship to attract females to enter a nest they guard or cuckoldry during which they steal fertilizations from a nest-holding male using a sneak or satellite spawning tactic, whereas type II males only cuckold. Using the neural activity marker phospho-S6, we show increased galanin neuron activation in courting type I males during mating that is not explained by their courtship vocalizations, parental care of eggs, or nest defense against cuckolders. This increase is not observed during mating in cuckolders of either morph or females (none of which show parental care). Together with their role in mating in male mammals, the results demonstrate an unexpectedly specific and deep-rooted, phylogenetically shared behavioral function for POA galanin neurons. The results also point to galanin-dependent circuitry as a potential substrate for the evolution of divergent phenotypes within one sex and provide new functional insights into how POA populations in teleosts compare to the POA and anterior hypothalamus of tetrapods.SIGNIFICANCE STATEMENT Studies of neuropeptide regulation of vertebrate social behavior have mainly focused on the vasopressin-oxytocin family. Recently, galanin has received attention as a regulator of social behavior largely because of studies demonstrating that galanin neurons in the preoptic area (POA) promote mating and parental care in mammals. Species with alternative reproductive tactics (ARTs) exhibit robust, consistent differences in behavioral phenotypes between individuals within a sex. Taking advantage of this trait, we show POA galanin neurons are specifically active during mating in one of two male reproductive tactics, but not other mating-related behaviors in a fish with ARTs. The results demonstrate a deep, phylogenetically shared role for POA galanin neurons in reproductive-related social behaviors with implications for the evolution of ARTs.

Keywords: alternative reproductive tactics; galanin; hypothalamus; preoptic area; reproduction; social behavior.

Figure 1.

Midshipman reproductive behavior and preoptic area. A, Line drawing of midshipman nest during mating. Courting type I male in nest with female laying eggs, and satellite mating male outside fanning sperm into the nest. Both type I and type II males satellite mate. B, Overhead drawing of midshipman brain. Arrows indicate level of sections shown in Ci and Cii. Scale bar, 1 mm. C, Nissl (cresyl violet)-stained coronal sections through midshipman brain at rostral (Ci) and caudal (Cii) levels of the preoptic area. Images in Figures 3–6 taken at similar level as Ci. C, Cerebellum; F, forebrain; H, hindbrain; M, midbrain; PM, magnocellular preoptic area; PPa, anterior parvocellular preoptic area; PPp, posterior parvocellular preoptic area; Tel, telencephalon.

Figure 2.

Timing of pS6 expression. A, pS6 expression is present 2 h after onset of humming behavior. Top, Timeline of humming behavioral experiment. Middle, Representative images of pS6 expression in VMN of humming (left) and control non-humming (right) animals collected 2 h after onset of humming fish's vocalization; animals from the courtship humming experiment (Fig. 4). Filled arrowheads indicate examples of strongly labeled VMN neurons in humming fish. pS6-labeled vocal pacemaker (premotor) neurons also visible immediately ventrolateral to VMN in humming male (3 somata in lower right corner). Bottom, Humming fish from the courtship humming experiment show a significant increase in the proportion of VMN neurons expressing pS6 2 h after the onset of humming, compared with non-humming control fish collected at the same time (t_(11.249) = −3.472, p = 0.005057, Welch two-sample t test). *p < 0.05. Error bars show mean (diamonds) ± SEM. B, pS6 expression persists at least 120 min after offset of humming behavior. Top, Timeline of post-humming isolation experiment. Bottom, Expression of pS6 is mostly absent in the VMN of male fish housed alone and then isolated for 30 min after humming (top left). Expression is, however, robust in fish isolated for 120 min after humming (top right). Filled arrowheads indicate examples of strongly labeled neurons. Expression is mostly absent in matched, non-humming control animals (bottom row). Open arrowhead in 120 min control image indicates imaging artifact. Scale bars. 100 μm.

Figure 3.

Mating behavior experiment. A, Divided tanks used for mating experiment (left) and close up image of type I male in an artificial nest with nearby hydrophone for sound recording (right) with experiment timeline (below). Photographs taken during lights on to enable easy visualization of setup. B, pS6 expression in galanin-expressing POA (POA^Gal) neurons of mating (left) and non-mating controls (right) for courting type I males, cuckolding type I males, cuckolding type II males, and females (non-mating control courting type I alone inside nest covered by mesh cage; control cuckolding type I and II males and females blocked from accessing a nest). Left images show galanin label in green, middle images show pS6 label in magenta, with merged images on the right. White arrowheads indicate location of galanin cell bodies. Scale bars, 50 μm. C, Proportion of POA^Gal neurons expressing pS6 in mating (blue) and control (red) courting type I males, cuckolding type I males, cuckolding type II males, and females. ANOVA F_(7,26) = 34.22, p = 1.5× 10⁻¹¹. *******p < 1× 10⁻⁷ Tukey's test. Error bars show mean (diamonds) ± SEM.

Figure 4.

Courtship humming behavioral experiment. A, Example of recording from a humming male (left) and a non-humming male (right) and timeline of experiment. B, pS6 expression in galanin-expressing POA (POA^Gal) neurons of humming and non-humming control animals. Left images show galanin label in green, middle images show pS6 label in magenta, with merged images on the right. White arrowheads indicate location of galanin cell bodies. Scale bars, 50 μm. C, Proportion of POA^Gal neurons expressing pS6 in humming (blue) and non-humming control (red) type I males (t_(7.2434) = −1.8073, p = 0.1122). Error bars show mean (diamonds) ± SEM. D, Relationship between pS6 expression in POA^Gal neurons and total hum duration during experiment for humming males (p = 0.3021, r = −0.418533). Inset, The proportion of POA^Gal neurons expressing pS6 is not correlated with length of time between end of humming and removal from nest for humming type I males. Pearson's correlation p = 0.2451, r = 0.465508. n.s., not significant.

Figure 5.

Egg care behavioral experiment. A, Experiment design. Example nests with eggs (left) and control nest without eggs (right) and timeline of experiment (below). Photographs taken during lights on to enable easy visualization of setup. B, pS6 expression in POA^Gal neurons of males in nests with eggs and control animals without eggs. Left images show Gal label in green, middle images show pS6 label in magenta, with merged images on the right. White arrowheads indicate location of galanin cell bodies. Scale bars, 50 μm. C, Proportion of POA^Gal neurons expressing pS6 in males in nests with eggs (blue) and control nests without eggs (red). Independent t test t₍₁₁₎ = 0.66927, p = 0.5171. No error bars shown due to extremely low variation within groups. Four of seven males in nests with eggs fanned eggs; all of these also brushed eggs. Two additional nesting males with eggs only brushed them. One male neither brushed nor fanned the eggs. Proportion of POA^Gal neurons expressing pS6 is not correlated with (D) the number of bouts of brushing eggs with mouth (Pearson's correlation p = 0.6706, r = −0.1978782). On the x-axis, two of the points overlap above 0 and two overlap to the right of 0 at 11 and 13 bouts, or (E) the amount of time spent fanning fins in the nest (Pearson's correlation p = 0.06185, r = 0.7312215). On the x-axis, four of the points overlap directly above 0; one of these is just to the right of 0 at 0.18 min fanning.

Figure 6.

Nest defense behavioral experiment. A, Timeline of experiment. B, pS6 expression in POA^Gal neurons of males mating with no cuckolders and with cuckolders present. Left images show galanin label in green, middle images show pS6 label in magenta, with merged images on the right. White arrowheads indicate location of galanin cell bodies. Scale bars, 50 μm. C, Proportion of POA^Gal neurons expressing pS6 in males mating without cuckolders (blue points) and with cuckolders present (red points). Blue brackets indicate the range of POA^Gal neurons expressing pS6 in courting type I males that mated in the earlier experiment (Fig. 3C). The red brackets indicate the range of POA^Gal neurons expressing pS6 in all other animals in all other experiments (Figs. 3C, 4C, 5C). No error bars shown due to small sample sizes.