Distribution and neuronal circuit of spexin 1/2 neurons in the zebrafish CNS

Kim, Eunmi; Jeong, Inyoung; Chung, Ah-Young; Kim, Suhyun; Kwon, Seung-Hae; Seong, Jae Young; Park, Hae-Chul

doi:10.1038/s41598-019-41431-7

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Published: 22 March 2019

Distribution and neuronal circuit of spexin 1/2 neurons in the zebrafish CNS

Eunmi Kim¹^na1,
Inyoung Jeong¹^na1,
Ah-Young Chung¹,
Suhyun Kim¹,
Seung-Hae Kwon²,
Jae Young Seong³ &
…
Hae-Chul Park ORCID: orcid.org/0000-0003-2095-3186¹

Scientific Reports volume 9, Article number: 5025 (2019) Cite this article

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Abstract

Spexin (SPX) is a highly conserved neuropeptide that is widely expressed in mammalian brain and peripheral tissue. In teleost, SPX1 is mainly expressed in the brain and ovary, and is involved in reproduction and food intake. A second form of SPX, SPX2, was recently identified in chick, Xenopus, and zebrafish. The expression pattern and roles of SPX2 are unknown. SPX (spx1) is highly expressed in the vertebrate brain, but its distribution, circuits, and interactions with its putative receptor are unknown. Here, we observed expression of spx1 in the midbrain and hindbrain, and spx2 in the hypothalamic preoptic area using in situ RNA hybridization in zebrafish. Analysis of transgenic reporter zebrafish revealed that hindbrain SPX1 neurons are PAX2⁺ inhibitory interneurons and project to the spinal cord, where they interact with galanin receptor 2b (GALR2b) neurons, suggesting that hindbrain SPX1 neurons are reticulospinal neurons. spx1 mRNA and SPX1 reporter expression were observed in dorsal habenula (dHb). SPX1 neurons in the dHb project to the interpeduncular nucleus (IPN), where GALR2a and GALR2b expression was also observed, suggesting that habenula SPX1 neurons may interact with GALR2a/2b in the IPN.

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Introduction

Neuropeptides play an important role in physiological functions, such as sleep, learning, memory, food intake, and regulation of body temperature. Neuropeptides act through a large variety of G-protein-coupled receptors (GPCRs) which are expressed on the cell surface, thereby contributing to neuronal communication. Spexin (also known as SPX, NPQ, and C12ORF39) is a secreted neuropeptide identified using bioinformatics^1,2. SPX consists of 14 amino acids that are highly conserved across vertebrate species, including human, rat, and goldfish. Previous reports have shown that the mammalian SPX is widely expressed in numerous tissues, including brain, pancreas, kidney^1,2,3, and adrenal gland⁴. RT-PCR analysis in teleosts such as goldfish and zebrafish has revealed that SPX (also known as SPX1) is expressed in the brain, ovary, liver, intestine, kidney, and heart^5,6.

The diversity of SPX expression suggests that SPX has multiple physiological functions. For example, SPX induces stomach muscle contraction¹ and inhibits adrenocortical cell proliferation⁴ in rat. In mice, SPX increases arterial pressure, decreases heart rate and urine flow, and has antinociceptive activity⁷. SPX is also associated with obesity and metabolism^8,9,10. In goldfish, SPX1 suppresses luteinizing hormone (LH) in the reproductive axis⁵ and reduces food consumption⁶. However, spx1 knock-out zebrafish show normal reproductive capability but higher food intake than that of wildtype fish, indicating that SPX1 is involved in the control of feeding but not reproduction in zebrafish¹¹. Recently, a second form of SPX, namely SPX2, has been identified in chick, Xenopus, and fish models using bioinformatics¹². However, the expression pattern and physiological roles of SPX2 have yet to be elucidated.

A recent ligand-receptor interaction study reported that SPX1/2 bind human galanin receptor type 2 (GALR2) and type 3 (GALR3), with higher potency toward GALR3 than that of galanin. Moreover, a recent study demonstrates that SPX, unlike galanin, is a biased agonist toward GALR2 preferring G-protein-mediated signalling over β-arrestin-dependent pathways, leading to signalling outputs which are different from those by galanin¹³. There are two forms of GALR2 in zebrafish, GALR2a and GALR2b; GALR3 has not been identified in this species. Zebrafish SPX1/2 also bind GALR2a/2b with higher potency toward GALR2b than that of galanin¹², indicating that SPX1/2 are functional agonists for zebrafish GALR2b. However, evidence for a direct interaction of SPX1/2 neurons with GALR2/3-epxressing cells in vivo has not been reported.

To assess the functions of SPX1/2 and interaction of SPX1 with its putative receptor, GALR2b, in zebrafish, we first investigated the expression of spx1 and spx2 in the developing and adult zebrafish central nervous system (CNS) by whole-mount in situ RNA hybridization. We next generated a transgenic reporter zebrafish line, Tg(spx1:mCherryCAAX) and Tg(spx1:gal4; uas:egfp), and analysed the distribution of SPX1 neurons and their interaction with GALR2b in the larvae and adult zebrafish CNS. Our findings provide evidence for potential functions of SPX1 and SPX2 in the zebrafish CNS.

Results

Distinct expression of spx1 and spx2 in the brain of zebrafish larvae

To examine the expression pattern of spx1 and spx2 during development, we first cloned spx1 and spx2 cDNA and analyzed their expression by RT-PCR. spx1 mRNA was first detected in one-cell stage zebrafish embryo but the expression faded, and then reappeared at 24 hpf. spx2 expression was detected from one-cell stage to 48 hpf continuously (Supplementary Fig. 1A). Whole-mount in situ RNA hybridization revealed that spx1 expression is first detected in a few cells in the hindbrain at 24 hpf (Supplementary Fig. 1B,C), and its expression was detected in the midbrain tegmentum and hindbrain of zebrafish larvae (Fig. 1A–D). However, we did not detect spx1 expression outside the brain. Conversely, spx2 mRNA was not detectable during development by whole-mount in situ RNA hybridization, but its expression was detected in the preoptic area of the hypothalamus of larval brain (Fig. 1E–G). We think that spx2 expression level is too low to be detected by whole-mount in situ RNA hybridization during development. The distinct expression pattern of spx1 and spx2 in the zebrafish brain suggests that the functions of different forms of SPX may be specialised or sub-functionalized in zebrafish. To confirm the expression of spx1 and spx2 in the hypothalamus, where SPX1 is involved in reproduction and feeding control in teleost^5,11, we performed fluorescent in situ RNA hybridization for spx1 or spx2 followed by immunohistochemistry with an antibody against α-melanocyte-stimulating hormone (α-MSH), a marker for the hypothalamus and pituitary gland¹⁴. We detected spx1 expression in the midbrain and hindbrain (Fig. 1H,I), but not in the hypothalamus (Fig. 1J). In contrast, spx2-expressing cells were observed in the preoptic area of anterior hypothalamus (Fig. 1K), rather than a-MSH expressing hypothalamus or pituitary (Fig. 1L), suggesting that SPX2 may be involved in neuroendocrine function.

To investigate the identity and neural circuitry of spx1-expressing cells in the zebrafish brain, we isolated ~3 kb of the 5′-upstream region of zebrafish spx1 from zebrafish genomic DNA by polymerase chain reaction (PCR) and established Tg(spx1:mCherry-CAAX) and Tg(spx1:gal4; uas:egfp) zebrafish which express mCherry-CAAX and EGFP fluorescent protein, respectively, in spx1-expressing neurons under the control of the spx1 promoter (Fig. 2 and Supplementary Fig. 2). Consistent with spx1 mRNA expression, mCherry fluorescence was detected in midbrain and caudal hindbrain neurons, and their nerve fibers in Tg(spx1:mCherry-CAAX) larvae (Fig. 2B,C). We also observed ascending projections from hindbrain SPX1 neurons to the midbrain area, descending projections to the spinal cord, and projections crossing the midline (Fig. 2B, arrows). Whole-mount in situ RNA hybridization for spx1 and mCherry mRNA revealed that mCherry was expressed in the midbrain and hindbrain in a pattern similar to that of the endogenous spx1 gene (Fig. 2D–I), suggesting that mCherry fluorescent protein expression was driven by spx1 regulatory elements.

To confirm SPX1/2 expression in the neuroendocrine system, we next examined mCherry and EGFP expression in Tg(spx1:mCherry-CAAX) and Tg(spx1:gal4; uas:egfp) larvae using antibodies against α-MSH, agouti-related protein (AgRP)¹⁴ and corticotropin-releasing hormone (CRH)¹⁵, markers for the hypothalamus and pituitary gland (Fig. 3). Tg(spx1:mCherry-CAAX) zebrafish were used to investigate axonal projections because mCherry-CAAX is localized in the membrane. Tg(spx1:gal4; uas:egfp) zebrafish were used to detect SPX1-expressing neuronal cell bodies (Supplementary Fig. 2). To detect spx2 expression, fluorescent in situ RNA hybridization was employed. Consistent with spx1 mRNA expression, spx1:mCherry⁺ and spx1:EGFP⁺ cells were not detectable in α-MSH⁺ (Fig. 3A,D), AgRP⁺ (Fig. 3B), or CRH⁺ (Fig. 3C) neurons in the hypothalamus and pituitary gland. Consistent with spx2 mRNA expression revealed by in situ RNA hybridization (Fig. 1E–G,K), a few spx2-expressing cells were detected in the preoptic area delineated by crhb expression (Fig. 3E, arrow).

Phenotypic and circuit-level characterisation of hindbrain SPX1 neurons

We next characterised the identity and neural connectivity of SPX1 neurons in the hindbrain. Approximately 20–30 spx1:mCherry⁺ cells were observed in the caudal hindbrain of Tg(spx1:mCherry-CAAX) zebrafish at 3 days post fertilisation (dpf) (Fig. 4A). Labelling of Tg(spx1:mCherry-CAAX) embryos with an anti-3A10 antibody, which marks Mauthner cells in rhombomere 4 of the hindbrain and their projections to the spinal cord¹⁶, indicated that spx1:mCherry⁺ cells were located in the caudal area of the hindbrain close to rhombomeres 6–7 (Fig. 4A). Labelling with an anti-HuC/D antibody revealed that hindbrain spx1:mCherry⁺ cells were HuC/D⁺ post-mitotic neurons (Fig. 4B). To characterise the neuronal subtype of hindbrain spx1:mCherry⁺ cells, we used Tg(isl1:gfp) zebrafish, which express GFP in cranial motor neurons¹⁷, and an anti-Pax2 antibody, which marks inhibitory interneurons^17,18,19. Analysis of the hindbrain of Tg(spx1:mCherry-CAAX); Tg(isl1:gfp) larvae revealed that hindbrain spx1:mCherry⁺ neurons were located near isl1:GFP⁺ cells but were not Islet1⁺ cranial motor neurons (Fig. 4C). Instead, labelling with an anti-Pax2 antibody showed that spx1:mCherry⁺ neurons were Pax2a⁺ interneurons (Fig. 4D,D’). To further examine the axonal projections of spx1:mCherry⁺ neurons in the hindbrain, we analysed mosaic expression of spx1:mCherry in the hindbrain of spx1:mCherry DNA-injected zebrafish larvae. We observed that hindbrain spx1:mCherry⁺ neurons had T-shaped axonal projections including ascending projections to the midbrain area, descending projections to the spinal cord, and projections crossing the midline (Supplementary Fig. 3).

Chx10-expressing V2a neurons in the hindbrain include reticulospinal neurons which have ipsilateral projections to the spinal cord and play critical roles in locomotor drive²⁰. Since hindbrain spx1:mCherry⁺ neurons had ipsilateral projections to the spinal cord (Fig. 2B and Supplementary Fig. 3), we next examined whether these neurons were also Chx10⁺ reticulospinal neurons using Tg(chx10:gfp) zebrafish, which express GFP in Chx10⁺ V2a neurons²¹. Analysis of Tg(spx1:mCherry-CAAX); Tg(chx10:gfp) larval hindbrain revealed that spx1:mCherry⁺ cells did not colocalise with medially located chx10:GFP⁺ V2a neurons. Instead, they were located in the lateral area of the hindbrain, separate to the Chx10⁺ neuronal cluster (Fig. 4E,E’). Zebrafish SPX1 binds to and activates galanin receptor 2a (GALR2a) and 2b (GALR2b), with higher potency toward GALR2b than that of galanin, suggesting that SPX1 is a functional agonist for GALR2b in zebrafish¹². Therefore, we next examined whether hindbrain SPX1 neurons interact with GALR2b neurons in the spinal cord using Tg(galr2b:egfp) zebrafish, which express EGFP in galr2b-expressing neurons²². Analysis of the spinal cord of Tg(spx1:mCherry-CAAX); Tg(galr2b:egfp) larvae revealed that hindbrain SPX1 neurons projected to the dorsal and ventral spinal cord, whereby dorsal projections were much thicker than ventral projections in the spinal cord (Fig. 4F,G’, arrows). Immunolabelling of Tg(spx1:mCherry-CAAX); Tg(galr2b:egfp) larval spinal cord with an anti-galanin antibody revealed that spx1:mCherry⁺ projections passed through the dorsal spinal cord and contacted with galr2b:EGFP⁺ neurons where galanin immunoreactivity was not detectable (Fig. 4G,G’). Galanin immunoreactivity was detected only in the white matter of the ventral spinal cord (Fig. 4G, yellow arrows), suggesting that GALR2b neurons in the dorsal spinal cord interact with hindbrain SPX1 neurons but ventral GALR2b neurons interact with galanin and/or SPX1.

We next examined whether spx1 was continuously expressed in the brainstem of adult zebrafish using whole-mount in situ RNA hybridization and confocal imaging of Tg(spx1:mCherry-CAAX) zebrafish (Fig. 5). Consistent with SPX1 expression in larvae, spx1 mRNA (Fig. 5A) and spx1:mCherry⁺ expression (Fig. 5B) were detected bilaterally in the brainstem of adult zebrafish. Immunolabelling with an anti-HuC/D antibody revealed that brainstem spx1:mCherry⁺ cells are HuC/D⁺ post-mitotic neurons (Fig. 5C). Transverse sections of the brainstem of adult Tg(spx1:mCherry-CAAX) zebrafish indicated that SPX1 neurons were located in the vagal motor nucleus and inferior reticular formation region (Fig. 5D,D’). We also observed SPX1 neuronal projections throughout the spinal cord of adult Tg(spx1:mCherry-CAAX) zebrafish, with enriched dorsal projections (Fig. 5E).

We next investigated whether hindbrain SPX1 neurons were excitatory or inhibitory by analysing their neurotransmitter content. Zebrafish hindbrain neurons have been shown to express various neurotransmitters including the vesicular glutamate transporter, vglut1, vglut2a, and vglut2b (markers for excitatory glutamatergic neurons); the neuronal glycine transporter, glyt2 (marker for inhibitory glycinergic neurons); and glutamic acid decarboxylase, gad65 (or gad2) and gad67 (or gad1b) (markers for inhibitory GABAergic neurons)²³. To investigate the neurotransmitter phenotype of hindbrain SPX1 neurons, we used Tg(vglut2a:gfp), Tg(gad1b:gfp)²⁴ and Tg(glyt2:gfp)²⁵ zebrafish which mark glutamatergic, GABAergic, and glycinergic neurons, respectively. Analysis of the hindbrain of double transgenic zebrafish larvae revealed that a subpopulation of spx1:mCherry⁺ neurons co-localised with gad1b:GFP (Fig. 6C,C’) but not vglut2a:GFP⁺ (Fig. 6A,A’) or glyt2:GFP⁺ neurons (Fig. 6B,B’). These findings suggested that a subpopulation of hindbrain SPX1 neurons was GABAergic.

Expression of spx1 and galr2a/2b in the habenula-interpeduncular nucleus circuitry

Galr2 and Galr3 knockout mice exhibit anxiety and depression-like phenotypes^26,27,28. Furthermore, GALR2 and GALR3 have been shown to be putative receptors for SPX in vitro¹², suggesting that SPX and GALR2/3 interaction may be involved in the regulation of anxiety and mood disorders. The medial habenula-interpeduncular nucleus (IPN) circuitry is implicated in anxiety and mood regulation. We thus investigated the expression of spx1/2, galr2a/2b, and galanin in the habenula and IPN. Whole-mount in situ RNA hybridization of the adult zebrafish brain for spx1, spx2, galr2a, and galr2b mRNA revealed that only spx1 was expressed in the dorsal habenula, which is the homologue of the medial habenula in mice (Fig. 7A, arrows), and galr2a/2b expression was detected in the ventral area of the IPN (Fig. 7B,C, arrowheads). To investigate in more detail the expression of SPX1/2 and GALR2a/2b in these regions, we used Tg(pou4f1-hsp70l:gfp) zebrafish, which express GFP in the medial subdivisions of the dorsal habenula (dHbM), which projects to the intermediate and ventral IPN²⁹. Consistent with the mRNA expression pattern, spx1:mCherry⁺ cells were first detected in a subpopulation of dorsal habenula neurons in 7 dpf larvae (Supplementary Fig. 4A) and its expression was also detected in adult Tg(spx1:mCherry-CAAX); Tg(pou4f1-hsp70l:gfp) zebrafish. However, only a few spx1:mCherry⁺ cells were co-localized with GFP⁺ cells (Fig. 7D,D’). Fluorescent in situ RNA hybridization with spx1 in the adult brain of Tg(narp:gal4vp16); Tg(uas:dsRed) zebrafish, which expresses DsRed in the lateral division of the dorsal habenula (dHbL), indicated that parts of spx1:mCherry⁺ cells were located in the dHbL (Supplementary Fig. 4B,B’). Notably, spx1:mCherry⁺ nerve fibers were detected in the dorsal IPN (dIPN), intermediate IPN (iIPN) and ventral IPN (vIPN) (Fig. 7G’,H”,I), suggesting that the IPN may receive input from spx1:mCherry⁺ cells in the dHb.

To examine whether SPX1 neurons in the Hb project to the IPN, we performed anterograde labelling in the habenula of adult Tg(spx1:mCherry-CAAX) zebrafish using neuronal tracer, DiI. We first confirmed that habenula cells were labeled with DiI (Fig. 7E) and found that dHbL cells co-localized with DiI and spx1:mCherry fluorescence (Fig. 7E’). In addition, we found that spx1:mCherry⁺ nerve fibers were co-localized with DiI-positive axon fibers in the fasciculus retroflexus (Fig. 7F,F’) and IPN (Fig. 7G,G’), suggesting that habenular SPX1 neurons project axons to the IPN. To further investigate the expression of galanin, SPX1, and GALR2b in the IPN, we labelled the brain of adult Tg(spx1:mCherry-CAAX); Tg(galr2b:egfp) zebrafish with an anti-galanin antibody. We detected galr2b:EGFP⁺ neurons throughout the entire IPN. spx1:mCherry⁺ nerve fibers were highly enriched in the i/vIPN but weak spx1:mCherry⁺ fluorescence was also detected in the dIPN. However, galanin immunoreactivity was rarely observed in the IPN region (Fig. 7H”’), suggesting that GALR2a and GALR2b cells located in the IPN may interact with SPX1 projections from the dHb. A high magnification confocal image of the IPN of adult Tg(spx1:mCherry-CAAX); Tg(galr2b:egfp) zebrafish showed that galr2b:EGFP⁺ neurons were in close contact with spx1:mCherry⁺ nerve fibers (Fig. 7I–I”). Collectively, these data suggest that nerve fibers from dHb SPX1 neurons interact with GALR2b neurons in the IPN, and SPX1-GALR2b interaction in the dHb-IPN circuitry may be involved in anxiety and mood regulation.

Discussion

In the present study, we describe the expression of SPX1 and SPX2 neuropeptides. We identify the phenotype and connectivity of SPX1 neurons and their interaction with GALR2b neurons in the zebrafish CNS. In mammals, SPX expression is widely expressed in various tissues including brain, skin, respiratory, digestive, urinary, and reproductive systems^1,2,3. In teleost, SPX1 is expressed mainly in the brain. The highest level of SPX1 expression was detected by RT-PCR in the goldfish brain including optic tectum, hypothalamus, and brainstem, with lower expression in the cerebellum, telencephalon, pituitary, liver, spleen, heart, intestine, and gonads^5,6. Zebrafish spx1 has mainly been detected in the brain and ovary by real-time PCR⁵ but spx2 expression has yet to be identified. In contrast to the broad expression of SPX in mammals, our data obtained by whole-mount in situ RNA hybridization and analysis of transgenic reporter zebrafish indicate that zebrafish spx1/2 expression is restricted in certain brain regions; spx1 in the midbrain and brainstem, and spx2 in the preoptic area of the hypothalamus. We did not detect spx1 expression in the hypothalamus, where SPX function is implicated in the regulation of reproduction and feeding control^5,6,11. Thus, SPX2 function may be involved in reproduction and feeding control in zebrafish. However, a recent study reported that spx1 knock-out zebrafish exhibited normal reproductive capability but higher food intake than wildtype fish, an effect mediated via increased expression of the appetite stimulant AgRP1¹¹. These findings suggest that SPX1 is involved in the control of feeding. Since SPX1 expression was not detectable in the hypothalamus, it appears that SPX1 neurons are not directly connected to appetite-regulating AgRP1 neurons, which are in the hypothalamus. We think that SPX1 neurons may be involved in the control the AgRP1 expression indirectly via other neural circuits.

We also observed approximately 20–30 SPX1 neurons bilaterally located in the caudal hindbrain of larval zebrafish and reticular formation region in the brainstem of adult zebrafish. Hindbrain SPX neurons are located in the caudal area near rhombomere 6–7 and have descending projections to the spinal cord, which are the characteristics of reticulospinal neurons. Reticulospinal neurons in the hindbrain play critical roles in providing descending excitatory input to the spinal cord locomotor systems in vertebrates^13,20,30,31. In zebrafish, sets of serially homologous reticulospinal neurons project to caudal levels of the spinal cord³², and T reticulospinal interneurons are located in the caudal area of the hindbrain with T-shaped axons including spinal projections³³. Based on the location in the caudal hindbrain and T-shaped axonal morphology, we speculated that hindbrain SPX1 neurons may be T reticulospinal interneurons. However, the axonal features of hindbrain SPX1 neurons differ slightly from T reticulospinal interneurons. In contrast to the spinal projections of T reticulospinal neurons which merge with Mauthner axons at the midline³³, nerve fibers of the hindbrain SPX1 neurons traversed the dorsolateral margin of the hindbrain and formed commissures in the spinal cord midline. Chx10⁺ V2a interneurons in the hindbrain include glutamatergic reticulospinal neurons which provide excitatory input for locomotor drive²⁰. However, hindbrain SPX1 neurons did not express Chx10, and they were GABAergic, suggesting that they provide inhibitory input to spinal cord neurons. These data indicate that although hindbrain SPX1 neurons have the phenotype of reticulospinal neurons which are located in the hindbrain and have spinal projections, they have different features to those of previously identified reticulospinal neurons. In addition, we observed that hindbrain SPX1 neurons interacted with dorsally located GALR2b neurons, which did not interact with galanin, in the spinal cord. Compared to galanin, SPX1 has greater potency for GALR2b in vitro¹². Collectively, our data suggest that SPX1 neurons in the hindbrain are reticulospinal neurons with spinal projections, and provide GABAergic inhibitory input for locomotor drive partly via interactions with GALR2b in the spinal cord. Consistent with our data, GABAergic reticulospinal interneurons have been reported to stop swimming in hatchling frog tadpoles by providing inhibitory input to locomotion³⁴.

The habenula is critical for behavioural choice and is involved in the regulation of pain, stress, fear/anxiety, sleep, and reward³⁵. The habenula consists of the medial and lateral habenula in mammals. The medial habenula mainly projects to the IPN, whereas the lateral habenula mainly projects to the ventral tegmental area and raphé³⁶. The IPN is a target of medial habenula neurons and projects to the raphé, which is part of the serotonergic system³⁷. The medial habenula is a component of the evolutionarily conserved dorsal diencephalic conduction system, which is essential for anxiety and mood regulation^38,39. In zebrafish, the dorsal habenula is the orthologue of the medial habenula in mice⁴⁰. Dorsal habenula-IPN circuitry is also implicated in fear and anxiety responses^41,42. In this study, we found that SPX1, but not SPX2, was expressed in the dHb in adult zebrafish, and its neuronal projections were connected to the IPN. Notably, we also found that galr2a and galr2b were expressed in the IPN, but galanin immunoreactivity was rarely detected in the habenula and IPN, suggesting that habenula SPX1 neurons may interact with GALR2a or GALR2b neurons in the IPN, and SPX1-GALR2a/2b neuronal circuits may be involved in regulating fear/anxiety responses. Our hypothesis is supported by previous studies, showing that GALR2 and GALR3 null mutant mice exhibit anxiety and depression-like phenotypes^26,27,28, while administration of SPX-based GALR2-specific agonist exerts an anxiolytic effect in mice⁴³. In addition, phylogeny and synteny analysis of the gene family has shown that zebrafish GALR2 is subdivided into GALR2a and GALR2b but it lacks GALR3, and zebrafish SPX1 can activate GALR2a and GALR2b in vitro whereby SPX1 has higher potency toward GALR2b than that of galanin¹². Collectively, our data suggest that, rather than galanin-GALR2a/2b interactions, habenula SPX1 neurons may interact with GALR2a or GALR2b in the IPN, and SPX1-GALR2a/2b neuronal circuits might be involved in the regulation of fear/anxiety responses.

Methods

Zebrafish lines

All experimental procedures were approved by the Korea University Institutional Animal Care& Use Committee (IACUC) and performed in accordance with Animal experiment guidelines of Korea National Veterinary Research and Quarantine Service. Wild-type AB strain zebrafish, Tg(spx1:mCherry-CAAX), Tg(spx1:gal4), Tg(uas:egfp), Tg(galr2b:egfp)²², Tg(isl1:gfp)¹⁷, Tg(chx10:gfp)²¹, Tg(vglut2a:gfp), Tg(gad1b:gfp)²⁴, Tg(glyt2a:gfp)²⁵, Tg(pou4f1-hsp70l:gfp)²⁹, and Tg(narp:gal4vp16)⁴¹, Tg(uas:dsred)⁴¹ zebrafish were used in this study. Adult zebrafish and zebrafish embryos were raised in 14 h light and 10 h dark cycle at 28.5 °C. Zebrafish embryos were staged according to days post-fertilisation (dpf), months post-fertilisation (mpf), and morphological characteristics. At 1 dpf, 0.003% (w/v) 1-phenyl-2-thiourea (PTU) in embryo medium was used for blocking pigmentation in zebrafish embryos. Also, 3~6-month-old male and female zebrafish were used in this study.

Cloning of spx1, spx2, galr2a, and galr2b

Total RNA was extracted using TRIzol reagent (Invitrogen) from wild-type strain AB embryos. The reverse transcription of RNA into cDNA was performed using ImProm-II™ Reverse Transcriptase (Promega), according to manufacturer’s instructions. To clone spx1, spx2, galr2a, galr2b, and corticotropin releasing hormone b (crhb), we designed PCR primers using sequence from a GenBank (spx1: XM_005164774.1, spx2: XM_005162991.1, galr2a: XM_002664007.3, galr2b: XM_001339133.3, crhb: NM_001007379.1) and amplified a product from 1 or 5 dpf cDNA. PCR products were cloned using pGEM-T easy vector (Promega).

Whole-mount in situ RNA hybridization and immunohistochemistry

Since SPX1/2 expression in larval brain and spinal cord (from 3 dpf to 7 dpf) is identical, we analysed the identity and circuit of SPX1 neurons in spx1:mCherry-CAAX transgenic fish at 3–4 dpf to obtain a whole brain image. For the analysis of SPX1/2 expression in the neuroendocrine system by in situ RNA hybridization and immunohistochemistry, we first isolated the whole brain from the skull of a 7 dpf larvae to improve penetration of probes/antibodies and to get the high-quality images. We used adult brain for the analysis of SPX1/2 expression in habenula because the expression of SPX1/2 in habenula is well detected in adult brain and not in the larval brain. For antisense RNA probe synthesis, the pGEM-T easy vector (Promega) containing spx1, spx2, galr2a, galr2b, crhb, gfp, and mCherry were linearised and transcribed by restriction endonucleases (NEB, New England Biolabs) and digoxigenin (DIG) labelling mixture (Roche). Wild-type AB and Tg(spx1:mCherry-CAAX) were fixed using 4% formaldehyde solution. spx1, spx2, galr2a, galr2b, crhb, gfp, and mCherry transcripts were detected in fixed zebrafish embryos and adult brains by whole-mount in situ RNA hybridization. Whole-mount in situ RNA hybridization was performed as described previously⁴⁴. For Immunohistochemistry, embryos and adult zebrafish were anesthetized by 200 mg/L of ethyl 3-aminobenzoate methanesulfonate salt (MS222, Sigma) until movement ceased and were then fixed in 4% paraformaldehyde. Fixed embryos were embedded in 1.5% agar blocks containing 5% sucrose and equilibrated in 30% sucrose solution. Frozen blocks were sliced into 10-µm sections using a cryostat microtome and mounted on glass slides. Sections were rinsed with PBS several times and then blocked in 2% bovine serum albumin with sheep serum. After the blocking reaction, sections were treated with primary antibodies for 2 hours at room temperature, washed for 2 hours with phosphate buffered saline (PBS), then treated with the appropriate secondary antibodies for 2 hours at room temperature. Images were obtained from the sections of Tg(spx1:mCherry-CAAX) and Tg(galr2b:egfp) zebrafish embryos. For immunohistochemistry, we used the following antibodies: mouse anti-HuC/D (1:200, Molecular Probes, Cat. No. A21271), mouse anti-3A10 (1:500, Developmental Studies Hybridoma Bank), rabbit anti-GAL (1:5000, Chemicon, Cat. No. AB5909), rabbit anti-alpha MSH (1:200, Phoenix Pharmaceuticals, INC, Cat. No. H-043-01), rabbit anti-AgRP (1:200, Phoenix Pharmaceuticals, INC, Cat. No. H-003-53), rabbit anti-CRH/CRF (1:500, Advanced Targeting Systems, Cat. No. AB-02), rabbit anti-Pax2 (1:200, Covance, Cat. No. PRB-276P), mouse anti-mCherry/Dsred (1:500, Clontech, Cat. No. 632392), rabbit anti-mCherry/Dsred (1:500, Clontech, Cat. No. 632496) and chicken anti-GFP (1:200, Abcam, Cat. No. AB13970). Alexa Fluor 488- and 568-conjugated secondary antibodies were used for fluorescent detection of primary antibodies (1:1000, Invitrogen, Cat. No. A-11001, A-11004, A11008, A-11011 and Abcam, Cat. No. ab96947).

Plasmid constructs and generation of transgenic zebrafish lines

To produce Tg(spx1:mCherry-CAAX) and Tg(spx1:gal4) zebrafish lines, approximately 3.2 kb fragment of zebrafish genomic DNA containing upstream sequences from the spx1 ATG start codon, was cloned into p5E-MCS⁴⁵. The following primer set was used for the PCR: forward primer (5′-CCCGACAGCTTTGCAGTATT-3′) and reverse primer (5′-AGCGGCAGGTTCCTCTTCAG-3′). The putative regulatory region was confirmed by sequence analysis. Next, we performed LR reaction with spx1-5′-, mCherryCAAX-middle- or gal4-middle- and polyA-3′-entry clones to generate spx1:mCherry-CAAX and spx1:gal4 DNA using LR II clonase (Invitrogen). To establish stable transgenic zebrafish lines, the spx1:mCherry-CAAX or spx1:gal4 DNA were injected into fertilised one-cell stage zebrafish embryos. To screen for germ-line transmitted transgenic zebrafish, we mated spx1:mCherry-CAAX DNA-injected founder zebrafish with wild-type zebrafish and screened the progeny for mCherry expression. We mated spx1:gal4 DNA-injected founder zebrafish with Tg(uas:egfp) zebrafish and screened the progeny for EGFP expression. The obtained transgenic founder fish were crossed with wild-type zebrafish, and F1 transgenic zebrafish were raised to adulthood.

DiI Labelling

We used the lipophilic tracer 1, 1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate (DiI, Invitrogen, D282) for neuronal tracing, similar to previous studies^29,46. We anesthetized the 6-12-month adult zebrafish by MS222 (Sigma) and performed transcardiac perfusion using PBS. The adult fish were then fixed in 4% paraformaldehyde with 0.25% glutaraldehyde (Sigma, G5882). Tiny crystals of the DiI were placed on the habenula of the fixed brains using a fine glass needle. The brains were rinsed by the identical fixative to detach extra dye particles, thereafter, the brains were incubated in the fixative at 37 °C for 3 days. Subsequently, the brains were embedded in 1.5% agar blocks containing 5% sucrose and equilibrated in 30% sucrose solution, followed by immunohistochemistry.

Data Availability

Data supporting the findings of this study are available in the article and its Supplementary Information Files, or from the corresponding authors on reasonable request.

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Acknowledgements

This work was supported by the Collaborative Genome Program of the Korea Institute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (MOF) (No. 20180430).

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Eunmi Kim and Inyoung Jeong contributed equally.

Authors and Affiliations

Department of Biomedical Sciences, Korea University, Ansan, Gyeonggido, 425-707, Republic of Korea
Eunmi Kim, Inyoung Jeong, Ah-Young Chung, Suhyun Kim & Hae-Chul Park
Korea Basic Science Institute, Chun-Cheon, Gangwon-Do, 24341, Republic of Korea
Seung-Hae Kwon
Department of Biomedical Sciences, Korea University, Seoul, 136-705, Republic of Korea
Jae Young Seong

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Eunmi Kim
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E.K., I.J. and H.C.P. designed the study, analyzed data, and wrote the manuscript. E.K., I.J., A.Y.C., S.K. and S.H.K. performed experiments. J.Y.S. advised and contributed to manuscript editing. All authors reviewed the manuscript.

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Kim, E., Jeong, I., Chung, AY. et al. Distribution and neuronal circuit of spexin 1/2 neurons in the zebrafish CNS. Sci Rep 9, 5025 (2019). https://doi.org/10.1038/s41598-019-41431-7

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Received: 10 October 2018
Accepted: 04 March 2019
Published: 22 March 2019
DOI: https://doi.org/10.1038/s41598-019-41431-7

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