Catecholaminergic Fiber Innervation of the Vocal Motor System Is Intrasexually Dimorphic in a Teleost with Alternative Reproductive Tactics

Abstract

Catecholamines, which include the neurotransmitters dopamine and noradrenaline, are known modulators of sensorimotor function, reproduction, and sexually motivated behaviors across vertebrates, including vocal-acoustic communication. Recently, we demonstrated robust catecholaminergic (CA) innervation throughout the vocal motor system in the plainfin midshipman fish Porichthys notatus, a seasonal breeding marine teleost that produces vocal signals for social communication. There are 2 distinct male reproductive morphs in this species: type I males establish nests and court females with a long-duration advertisement call, while type II males sneak spawn to steal fertilizations from type I males. Like females, type II males can only produce brief, agonistic, grunt type vocalizations. Here, we tested the hypothesis that intrasexual differences in the number of CA neurons and their fiber innervation patterns throughout the vocal motor pathway may provide neural substrates underlying divergence in reproductive behavior between morphs. We employed immunofluorescence (-ir) histochemistry to measure tyrosine hydroxylase (TH; a rate-limiting enzyme in catecholamine synthesis) neuron numbers in several forebrain and hindbrain nuclei as well as TH-ir fiber innervation throughout the vocal pathway in type I and type II males collected from nests during the summer reproductive season. After controlling for differences in body size, only one group of CA neurons displayed an unequivocal difference between male morphs: the extraventricular vagal-associated TH-ir neurons, located just lateral to the dimorphic vocal motor nucleus (VMN), were significantly greater in number in type II males. In addition, type II males exhibited greater TH-ir fiber density within the VMN and greater numbers of TH-ir varicosities with putative contacts on vocal motor neurons. This strong inverse relationship between the predominant vocal morphotype and the CA innervation of vocal motor neurons suggests that catecholamines may function to inhibit vocal output in midshipman. These findings support catecholamines as direct modulators of vocal behavior, and differential CA input appears reflective of social and reproductive behavioral divergence between male midshipman morphs.

Figure 1

Intrasexual morphometric comparison of forebrain T H-ir nuclei. Micrographs depict TH-ir neurons (arrowheads) in representative transverse sections through the anterior parvocellular preoptic nucleus (PPa) (a) and periventricular posterior tuberculum (TPp) (c) in a type I male. Right edge of (a) is at the midline, (c) is centered on the midline. MFB = medial forebrain bundle, PVO = paraventricular organ. Scatter dot plots in (b) and (d) represent mean ± SE number of total TH-ir neurons. Scale bar = 100 µm.

Figure 2

Intrasexual morphometric comparison of hindbrain TH-ir nuclei. Micrographs depict TH-ir neurons (arrowheads) in representative transverse sections through the locus coeruleus (LC) (a), vagal lobe (XL) (c), and area postrema (AP) (e) in a type II male. Right edges of (a) and (c) are at the midline of the brain, (e) is centered on the midline. Inset in (c) depicts larger extraventricular vagal neurons lying caudal and ventrolateral to smaller paraventricular neurons in XL. Scatter dot plots in (b), (d), and (f) represent mean ± SE number of total TH-ir neurons. *p = 0.0123. Scale bar = 100 µm.

Figure 3

Intrasexual morphometric analysis of the relationship between total TH-ir cell number and body size in the hindbrain. Pearson correlations of total TH-ir cell number scaled to standard length in the vagal-associated nuclei (paraventricular and extraventricular cells combined) (a) and area postrema (AP) (c). Closed circles represent type I males, open circles are type II males. Scatter dot plots in (b) and (d) represent mean ± SE number of TH-ir neurons corrected for body size. *p = 0.0219. See Table 2 for statistical breakdown of TH-ir vagal subgroups.

Figure 4

Micrographs showing representative transverse sections through the hypothalamic and midbrain components of the vocal-motor pathway in a type II male. Anterior tuberal hypothalamus (AT) (a), ventral tuberal hypothalamus (vT) (b), midbrain periaqueductal grey (PAG) (c), and paratoral tegmentum (PTT) (d). Compass in bottom right corner of (a) indicates anatomical orientation across all images: D = dorsal; V = ventral; M = medial; L = lateral. ca = cerebral aqueduct, Pe = periventricular cell layer of TS = torus semicircularis. Mean ± SE TH-ir fiber density for type I vs. type II males: AT (7.37 ± 1.28% vs. 9.1 ± 1.19%); vT (2.59 ± 0.12% vs. 3.1 ± 0.25%); PAG (1.9 ± 0.28% vs. 1.84 ± 0.31%); PTT (7.51 ± 0.82% vs. 7.96 ± 0.89%). Scale bar = 100 µm.

Figure 5

Intrasexual morphometric comparison of TH-ir fiber density within the vocal motor nucleus (VMN). Micrographs depict representative transverse sections through the VMN at low and high magnifications in a type I (a, c) and type II (b, d) male, Arrowheads in c and d indicate punctate-type contacts on individual motor neurons. Scatter dot plots in (e) represent mean ± SE TH-ir fiber density per fractional area (5,692.7 µm²); (f) are mean ± SE number of putative TH-ir contacts per cell. ****p < 0.0001; **p = 0.0059. Scale bar in (a) and (b) = 100 µm, scale bar in (c) and (d) = 35 µm.