Galanin immunoreactivity is sexually polymorphic in neuroendocrine and vocal-acoustic systems in a teleost fish

Abstract

Galanin is a peptide that regulates pituitary hormone release, feeding, and reproductive and parental care behaviors. In teleost fish, increased galanin expression is associated with territorial, reproductively active males. Prior transcriptome studies of the plainfin midshipman (Porichthys notatus), a highly vocal teleost fish with two male morphs that follow alternative reproductive tactics, show that galanin is upregulated in the preoptic area-anterior hypothalamus (POA-AH) of nest-holding, courting type I males during spawning compared to cuckolding type II males. Here, we investigate possible differences in galanin immunoreactivity in the brain of both male morphs and females with a focus on vocal-acoustic and neuroendocrine networks. We find that females differ dramatically from both male morphs in the number of galanin-expressing somata and in the distribution of fibers, especially in brainstem vocal-acoustic nuclei and other sensory integration sites that also differ, though less extensively, between the male morphs. Double labeling shows that primarily separate populations of POA-AH neurons express galanin and the nonapeptides arginine-vasotocin or isotocin, homologues of mammalian arginine vasopressin and oxytocin that are broadly implicated in neural mechanisms of vertebrate social behavior including morph-specific actions on vocal neurophysiology in midshipman. Finally, we report a small population of POA-AH neurons that coexpress galanin and the neurotransmitter γ-aminobutyric acid. Together, the results indicate that galanin neurons in midshipman fish likely modulate brain activity at a broad scale, including targeted effects on vocal motor, sensory and neuroendocrine systems; are unique from nonapeptide-expressing populations; and play a role in male-specific behaviors.

Keywords: RRID:AB_142018; RRID:AB_2157629; RRID:AB_2534017; RRID:AB_2534018; RRID:AB_2783794; RRID:AB_477652; RRID:AB_90782; alternative reproductive tactics; auditory; galanin; neuroendocrine; preoptic area; vocal.

FIGURE 1

Midshipman vocal-acoustic network. (a) Line drawing in sagittal plane of the plainfin midshipman (Porichthys notatus) brain with relative regions of major vocal and auditory regions outlined (modified from Bass & McKibben, 2003; Kittelberger, Land, & Bass, 2006; Pengra, Marchaterre, & Bass, 2018). Shaded regions contain Gal-ir fibers in all morphs. Un-shaded regions contain Gal-ir fibers in males that are greatly reduced or absent in females. Black dots indicate presence of Gal-ir somata in the region. Red lines show connections of the vocal motor system and blue lines indicate connections of the auditory system with arrowheads showing direction of connection. Connections without arrowheads are reciprocal. AudTeg is auditory tegmentum, including paralemniscal tegmentum, nucleus of the lateral lemniscus, and isthmal nucleus (see text for details). Vertical lines indicate approximate level of low magnification images in Figures 2, 3, and 4. Figure 2h, a section through the pituitary, is at the level of Figure 3a. The levels shown for Figures 2c, 3e, and 4e correspond to the levels shown in Figure 5 for the POA-AH (5a–f), PAG (5g–i), and VMN (5j–l), respectively. (b) Nissl-stained coronal sections through a nonexperimental type I male midshipman brain at the approximate levels shown in Figures 2–4. Labels above sections indicate the figure number and panel that correspond to that section. Also see list of abbreviations

FIGURE 2

Forebrain galanin (Gal) distribution. Gal-immunoreactivity (green) in the telencephalon and preoptic area of the midshipman brain with DAPI nuclear stain (blue). Images from a representative type I male taken at the approximate levels indicated in Figure 1a. Also see Figure 1b for representative Nissl-stained sections from nonexperimental material that provide cytoarchitectural detail for illustrated regions. (a) Gal label appears in moderate density beginning in dorsal (Vd) and ventral (Vv) nuclei of the ventral telencephalon. Box indicates region shown in (d). (b) Density of Gal label increases at the level of the anterior commissure (ac), where the anterior parvocellular nucleus of the preoptic area (PPa) begins; sparse fibers also appear in dorsal telencephalon. Box indicates region shown in (e). (c) Gal-ir somata are most abundant in PPa. Box indicates region shown at higher magnification in (f), arrows indicate Gal-ir somata. (g) Somata are also present throughout the posterior parvocellular nucleus of the preoptic area PP_p, small cells), which runs lateral and ventral to the magnocellular neurons within the magnocellular nucleus of the preoptic area (PMm, larger cells) at this level. Box in (g) shows region containing Gal-ir cells in PPp shown at higher magnification in (j). Lines indicate dorsal-ventral extent of PMm and PPp. (h) Gal-ir fibers in the pituitary (i) Gal-ir somata continue into magnocellular preoptic area (PM). Only PMm cells express Gal, though they are interspersed with even larger gigantocellular neurons within the PM (PMg). Fibers are sparse within PM, but dense fibers are present lateral to PM and in the preoptico-hypophyseal tract (PHT). Box indicates region depicted at higher magnification in (k). (k) Gal-ir PMm neurons. Letter g indicates a gigantocellular cell that is not labeled for Gal. Also see list of abbreviations

FIGURE 3

Diencephalon and midbrain galanin (Gal) expression. Gal-immunoreactivity (green) in the diencephalon and midbrain of the midshipman with DAPI nuclear stain (blue). Images from a representative type I male taken at the approximate levels indicated in Figure 1a. Also see Figure 1b for representative Nissl-stained sections from nonexperimental material that provide cytoarchitectural detail for illustrated regions. (a) Fibers present in several diencephalic nuclei, including sparse fibers in rostal end of anterior tuberal nucleus (AT) and denser fibers within pretectal nuclei (PPd/PPv), central posterior (CP), and periventricular nucleus of the posterior tuberculum (TPp). (b) Fibers continue more caudally, and sparse fibers appear at rostral level of lateral hypothalamus (LH). (c) Gal-ir neuron in AT at similar level as (b). (d) Fiber densities at the level of anterior midbrain. Fibers apparent in stratum album centrale (SAC) and stratum griseum centrale (SGC) layers of the mesencephalic tectum (TeM) and rostralmost level of torus semicircularis (TS) and periaqueductal gray (PAG). Fiber density highest in AT and LH at this level. (e) Gal-ir neuron in LH at similar level as (d). (f) Continuing fiber label in the midbrain including tectum (TeM), periaqueductal gray (PAG) and torus semicircularis (TSp/TSd), and caudal diencephalon. Boxes indicate regions shown at higher magnification in (g) and (h) for TeM and PAG, respectively. (g) Gal-ir fibers present in SAC and SGC layers of TeM. (h) PAG contains some of the most dense Gal-ir fibers in the male brain. (i) Gal-ir fibers continue through the caudal midbrain, including nuclei of the auditory tegmentum: Paralemniscal nucleus (PL), nucleus of the lateral lemniscus (nLL), and isthmal auditory-vocal nucleus (IS). Also see list of abbreviations

FIGURE 4

Hindbrain galanin (Gal) expression. Gal-immunoreactivity (green) in the hindbrain of the midshipman with DAPI nuclear stain (blue). Images from representative type I males taken at the levels indicated in Figure 1a. Also see Figure 1b for representative Nissl-stained sections from non-experimental material that provide cytoarchitectural detail for illustrated regions. (a) Sparse Gal-ir fibers in the granule cell layer of the cerebellum (Cg) with dense fibers throughout the reticular formation (RF). Box indicates region shown at higher magnification in insert. (b) Fibers in octaval efferent nucleus (OEN) (higher magnification in lower insert indicated by box); label is dense at this level in rostral-intermediate division of the descending octaval nucleus (DOri, higher magnification in upper insert indicated by box). Fibers are found in the vocal-motor central pattern generator of males only, including vocal prepacemaker (VPP) (c,d), pacemaker (VPN) (e,g), and vocal motor (VMN) nuclei (e,f). Fibers are sparse within nuclei, but highly dense in the neuropil immediately surrounding VPP, and VPN/VMN. Boxes in (c) and (e) indicate regions shown at higher magnification in (d), (f), and (g). Images in (c–g) taken from a fish collected following humming and colabeled with phosphorylated S6 protein, a neural activity marker (Knight et al., 2012), which positively labeled and aided identification of VPP and VPN (J. A.Tripp, I. Salas-Allende & A. H. Bass; unpublished observations). Also see list of abbreviations

FIGURE 5

Sex and male morph differences in galanin (Gal)-ir distribution in the midshipman brain. Gal-immunoreactivity (green) with DAPI nuclear stain (blue). Gal-ir neurons found in anterior parvocellular preoptic area (PPa) of all morphs (a–f), but much less abundant in females (c, f). Males also have much more dense expression of Gal-ir fibers in midbrain tectum (TeM), periaqueductal gray (PAG), and torus semicircularis (TS) (g, h) compared to females (i); type II label is also reduced compared to type I males. In the hindbrain, type I males have sparse Gal-ir fibers within the pacemaker (VPN) and vocal motor (VMN) nuclei and dense expression surrounding these nuclei (j), which is reduced in type II males (k). Females show no Gal-ir fibers within VPN or VMN and greatly reduced expression of fibers in the surrounding hindbrain compared to males (l). Boxes in (a–c) indicate regions shown at higher magnification in (d–f) and boxes in (g–l) indicate regions shown in inserts. Also see list of abbreviations

FIGURE 6

Male midshipman have significantly more galanin (Gal) neurons in the preoptic area-anterior hypothalamus than females. Violin plots showing the total number of Gal-ir neurons in the preoptic area of type I male, type II male, and female midshipman after Abercrombie correction (Abercrombie, 1946) and adjustment for standard length. Width of each plot represents the probability density of the data. Lower and upper bounds of boxes indicate first and third quartiles, thick line within the box indicates median value, and whiskers extend to minimum and maximum values. Dots represent individual data points from each animal. One way ANOVA F_(2,15) = 4.937, p = .0225. Groups labeled with different letters significantly differed from one another by Tukey post hoc comparisons. ANOVA, analysis of variance

FIGURE 7

Neuropeptide coexpression with galanin (Gal) in the preoptic area-anterior hypothalamus of midshipman. (a–c) Galanin neurons do not coexpress arginine vasotocin (AVT) as documented using an antibody to its mammalian homologue, arginine vasopressin (AVP): (a) Gal-immunoreactive (ir) label (green), (b) AVP-ir label (magenta), (c) merged images showing that Gal and AVP/AVT are expressed in different cells. Image is from a type I male brain between the levels of Figure 2c,g. Arrows indicate position of Gal-ir somata here and in (d–i). (d–f) Very few galanin (Gal) neurons coexpress isotocin (IT) as documented using an antibody to its mammalian homologue, oxytocin (OT): (d) Gal-ir label (green), (e) OT-ir label (magenta), (f) merged images showing that Gal and OT/IT are expressed in different cells. Image is from a type I male brain at a similar level as Figure 2c. Arrows indicate position of Gal-ir somata. Insert in (f) shows rare example of Gal-ir neuron with OT-ir colabel (hollow arrow), surrounded by Gal-ir neurons that lack colabel (solid arrows). Insert image taken from type I male. In contrast to AVP/AVT and OT/IT, approximately 20–25% of Gal neurons also express GABA in both male morphs and females. Example image containing two Gal+/GABA+ neurons (upper two) and two Gal+/GABA− neurons (lower two). (g) Gal-ir label (green), (h) GABA-ir label (magenta), and (i) merged image. Image is from a type I male brain at similar level as Figure 2i