A fundamental aspect of studying animal physiology and behavior is understanding the pathways and mechanisms by which they are regulated. Many studies have focused on understanding how certain neurochemicals, such as neurotransmitters or neuromodulators, influence behavior. One such family of neurochemicals, a class of nine amino acid molecules known as nonapeptides, is of particular interest. Nonapeptides are highly conserved across vertebrates and play crucial roles in numerous physiological functions and behaviors (1). Their exact effects vary widely between species for reasons that are not fully clear, making them the subject of studies spanning taxa, sexes, social contexts, brain regions, and scientific fields.

One of the nonapeptides, arginine vasopressin (AVP; also known as antidiuretic hormone, ADH), is of particular interest in the study of social behavior across animals. AVP is a highly conserved nonapeptide that has a wide range of modulatory effects across vertebrates (2). Most vertebrate classes possess the ancestral nine amino acid peptide form, arginine vasotocin (AVT; AVP has a phenylalanine substitution of isoleucine in position 3) (3). Originally identified for its role in osmoregulation, cardiovascular function, and stress hormone release (4–6), AVP/T has also been shown to play a key role in modulating social behavior such as courtship and aggressive behavior in fish (7–9), amphibians (10–12), birds (13–16), and in mammals (17–19). AVP/T has also been shown to modulate territoriality and space use [reviewed in Ref. (20)] and alternative reproductive phenotypes in teleost fish (21–29). These effects are mediated by sex, social context, and the neural expression of the nonapeptide and its receptors (2, 3).

AVP/T is synthesized in magnocellular neurons of the hypothalamus in animals and is produced from prohormones that also encode a carrier protein, neurophysin. There are two types of neurophysin: the prohormone proxyphysin that is hydrolyzed to oxytocin and neurophysin I, and the prohormone propressophysin that is hydrolyzed to vasopressin and neurophysin II, in addition to a short glycopeptide (Figure 1). Studies previously done in mammals have shown that these distinct neurophysins may be essential for the implementation of hormonal activity (30). The axon terminals of these hypothalamic neurons extend to the neurohypophysis, where the secretions of these neurosecretory cells are picked up by the circulatory system and transported to target organs.

In the brain, AVP/T exerts its effects in particular regions by binding to distinct receptors. The expression of these receptors differs across tissues and by function (33, 34). For example, the AVP/T receptor subtype, V1a, has been shown to regulate sex and species differences in many social behaviors in mammals, birds, amphibians, and fish (24, 35–37). For teleosts in particular, AVT receptors consist of one V2-type and two V1a types (V1a1 and V1a2) (38–40). The distributions of these receptors are widespread throughout the brain and are found in regions of interest for social regulation, such as the olfactory bulb (OB), telencephalic areas, POA, hypothalamus, midbrain sensory regions, and hindbrain regions important for social approach responses (41, 42).

AVP/T cell bodies are found in the preoptic area-anterior hypothalamus (POA-AH) complex, an integration center that also regulates numerous physiological and hormonal processes through the pituitary gland (16, 23, 43–48). AVP/T peptides are produced by populations of magnocellular and parvocellular neurons within this POA-AH complex. In amniotes, these magnocellular neurons are found in the supraoptic nucleus (SON) of the hypothalamus, while parvocellular neuron populations are found in the paraventricular nucleus (PVN) of the hypothalamus (3, 47). In fish and amphibians, AVT in these magnocellular and parvocellular neuronal populations are found in the POA and AH. These cell groups project to the neurohypophysis, where AVP/T exerts a wide range of peripheral effects (31). Previous studies have used immunohistochemical (IHC) techniques to label immunoreactivity of AVP/T protein product, or in situ hybridization (ISH) to label AVP/T preprohormone mRNA across the brain. Table 1 provides a summary of the brain regions where AVP/T has been found, along with the technique used to map either AVP/T protein product or label AVP/T preprohormone mRNA in the respective studies. In general, amniotes have similar patterns of AVT expression throughout the forebrain. In teleosts, however, AVT-containing neurons have been shown to be localized to the POA region.

Tetrapod vertebrates exhibit additional anatomical characteristics that remain largely conserved. AVP is produced in neurons of the bed nucleus of the stria terminalis and the medial amygdala, and projections extend to the lateral septum, nucleus accumbens, amygdala, and periaqueductal gray (PAG) (47, 103, 104). These circuits are particularly important for social behavior, such as mate affiliation, nest defense, and parental care of offspring (92, 105–107). Putative teleost homologs of these regions also contain AVT fiber innervation, though these fibers are generally thought to originate in the POA (22, 55). AVP/T fibers are located throughout the brain in jawed vertebrates, likely conserved for at least 500 million years, including the POA, anterior and lateral hypothalamic areas, midbrain tegmentum, PAG, isthmal structures (i.e., locus coeruleus), and viscerosensory areas of the caudal medulla (3).

In the teleost POA, the magnocellular and gigantocellular AVT neuron populations are hypothesized to be homologous to the supraoptic nucleus in tetrapods based on colocalization with corticotropin-releasing hormone-producing neurons and expression of the Nurr1 receptor, while the parvocellular cell group is the putative homolog of the PVN of the mammalian POA (47, 49, 108, 109). AVT appears to be limited to the POA (1). Weaker expression also appears in the anterior tuberal nucleus of the hypothalamus [aTn; (21, 23)], the putative teleost homolog of the mammalian ventromedial hypothalamus [VMH; (110, 111)]. As in tetrapods, AVT is found in the parvocellular, magnocellular, and gigantocellular neuron groups, which are distinguished by soma size and location, with gigantocellular populations being found most caudally. These AVT neurons have been shown to project to the posterior pituitary through the preoptico-hypophysial tract as well as various regions in the ventral telencephalon and ventral thalamus (23, 112). Overall, the expression of AVT preprohormone mRNA and peptide seems to be fairly conserved across vertebrates. There might be an ascertainment bias as most studies only report on the POA and/or used IHC methods to map AVT-positive neurons, which may not be sensitive enough to detect low levels of peptide expression in other brain regions [but see Ref. (21, 51, 59, 113)].

Importantly, AVP/T has been shown to be socially regulated [see Ref. (3, 20) for reviews]. For example, non-monogamous male Montane voles have fewer V1a receptors in the ventral pallidum compared to monogamous Prairie voles, and the induction of these receptors in the Montane voles via viral vector gene transfer yields pair bonding behavior similar to Prairie voles (114). White-throated male sparrows (Zonotrichia albicollis) have more AVT expression in the medial portion of the BNST and in a subdivision of the caudal lateral septum compared to tan-striped male sparrows. This neural AVT expression is associated with aggression, since white-striped males defend their territories more vigorously and intrude into other territories more often than their tan-striped male counterparts (115). Research in teleosts suggests that AVT preprohormone mRNA levels might be more reliable indicators of social status than the number or size of AVT-positive neurons (as determined by immunohistochemistry). In Burton’s Mouthbrooder cichlid, Astatotilapia burtoni, socially dominant males exhibit higher levels of AVT expression than subordinate males in gigantocellular nucleus of the preoptic area, whereas the inverse was found in the parvocellular preoptic nucleus (21). The number or size of AVT-immune-reactive (ir) neurons was, however, not correlated with behavior (126). Similarly, in the sex-changing Bluehead wrasse, Thalassoma bifasciatum, preoptic AVT mRNA levels predicts male behavior robustly, while AVT-ir neuron size does not (59). These examples illustrate the role AVP/T plays in modulating social behavior across species, and how these effects are not just sex- and context-specific but also brain region-specific.

The majority of studies that examine the expression and distribution of either AVT preprohormone mRNA or the AVT peptide in teleost fish have primarily focused on the POA. These studies utilize quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry, immunocytochemistry, or radioactive ISH to quantify mRNA and/or protein expression (for more information regarding these methods see Table 2). In the present study, we revisit the neural distribution of AVT nonapeptide expression, in particular expanding on the existing knowledge of its mRNA distribution within the forebrain of a highly social cichlid fish. We first used ISH to examine whether the AVT preprohormone mRNA is expressed in pallial and subpallial regions of the telencephalon of A. burtoni. In a second experiment, we used qPCR to ask whether AVT preprohormone mRNA expression in pallial area Dm, the putative homolog of the mammalian basolateral amygdala, is modulated by social context. We provide evidence of AVT preprohormone mRNA expression in forebrain regions never previously reported to contain nonapeptides in teleost fish. Furthermore, our results suggest that AVT preprohormone mRNA expression in the putative homolog of the mammalian basolateral amygdala can be regulated by social context.

In the present study, we have shown that expression of AVT preprohormone mRNA in the cichlid fish A. burtoni is not limited to preoptic nuclei and the anterior tuberal nucleus. Rather, AVT preprohormone mRNA is expressed widely throughout pallial and subpallial regions not previously associated with the expression of the AVT nonapeptide. We have also found evidence for the social regulation of AVT expression within area Dm-1, the putative homolog of the mammalian basolateral amygdala. These surprising findings provide an important addition to our understanding of the distribution of AVT in the teleost brain and how nonapeptides modulate social behavior in cichlids.

Previous studies in teleost fish have reported the presence of AVT preprohormone and AVT peptide primarily in the POA and the aTn of the hypothalamus (21, 59, 121). Several studies also mapped AVT-immunoreactive fibers and found that they project extensively throughout the teleost brain, although where these fibers originate is not always obvious (55, 121). Our data expand on these studies to show the expression of AVT preprohormone mRNA in multiple regions of the dorsal, medial, central, and ventral pallium. Specifically, subpallial regions, such as the medial and lateral divisions of area Vs [putative homolog of the medial amygdala and the bed nucleus of the stria terminalis (109)] along with area Vv (putative septum homolog) and the central part of area Vd (putative striatum homolog) showed robust expression of AVT preprohormone mRNA, while pallial regions, including basolateral amygdala (area Dm) and hippocampus (area Dl), showed less but still reliably detectable abundance. Our qPCR results confirm expression of AVT in area Dm, and we show this expression to be modulated by the social context. These results suggest that AVT expression in teleosts may be more similar to AVP/T expression in birds and mammals.

If there are indeed AVT expressing neurons in the teleost pallium, why did previous authors fail to detect them? First, methodological limitations may provide an answer: all studies examining the expression and distribution of either AVT preprohormone mRNA or the AVT peptide in teleost fish to date utilize IHC, qPCR, or radioactive ISH to detect peptide and/or mRNA expression (see Table 1). Most do not provide information on telencephalic brain regions, instead focusing exclusively on the preoptic AVT cell populations. The few studies that investigate whether AVT preprohormone or peptide is present in the teleost telencephalon and other areas outside the preoptic nuclei and hypothalamus (21, 51), rely on either radioactive ISH or qPCR of the entire forebrain. Importantly, it is well understood that the former requires short exposure times so as to not overdevelop the signal in preoptic AVT neurons, where the preprohormone is expressed at very high levels [see, e.g., Ref. (21, 59)].

Second, it is also conceivable that AVT transcripts are transported from preoptic cell bodies to fibers (putative axons) in various telencephalic regions for local synthesis (possibly near varicosities or putative release sites). Using both ISH and PCR (122), found oxytocin preprohormone mRNA in axons and Herring bodies in the lateral and ventral hypothalamus, the median eminence, and the posterior lobe of the pituitary in rats. While it is unclear whether this can also occur in axons projecting into the telencephalon, these results nevertheless indicate that at least in the rodent oxytocin preprohormone mRNA can be transported axonally. Given that (a) oxytocin and AVP/T genes as paralogs may share a similar molecular and cellular machinery, and (b) teleosts have brain regions putatively homologous to these rodent regions (109, 117), the signal we detect in pallial regions may indeed be the consequence of axonal transport of AVP/T mRNA. Given the ISH methods used in this study, we cannot conclusively deduce if the mRNA signal resembles varicosities or puncta. Detailed tract tracing studies in combination with sensitive assays such as ISH will allow us to test this hypothesis.

Finally, another possible explanation for the distribution of AVT mRNA expression throughout the teleost telencephalon could be that we are observing preprohormone mRNA that never is translated and processed into the mature peptides AVT and/or neurophysin II. Although the enzymes processing preprohormones could be present in putative pallial AVT neurons for processing peptides others than AVT and neurophysin, any future analysis (e.g., by ISH) demonstrating that these enzymes do not co-localize in these neurons would support this idea. Alternatively, only neurophysin might be produced, for a yet to be discovered function, which can be tested once a specific antibody is available. These possible explanations notwithstanding, our results should be seen as an encouragement to examine telencephalic AVT expression in a range of teleost species.

Is telencephalic AVT of functional importance in A. burtoni? Interestingly, we did find significant variation in AVT preprohormone mRNA levels, albeit lowly abundant, depending on social context in area Dm-1, the putative homolog of the mammalian basolateral amygdala (109). This region is known to be important for fear conditioning in mammals, as well as being a sensory integration center that mediates emotional behavior (123, 124). Here, AVT shows increased relative expression in dominant males in the presence of a familiar neighbor, which has important implications for territory defense (125). A possible explanation for this result is that AVT expression in the Dm-1 may be modulating an individual’s behavioral response to a familiar neighbor, possibly facilitating social habituation. It is important to note that we do not know baseline AVT levels in the Dm-1, and the data only represent expression in response to an intruder in a joint defense paradigm (118). Further support for a functional role of AVT expression in the basolateral amygdala homolog is provided by the finding that other candidate genes followed the same expression pattern across experimental groups that we observed with AVT expression, possibly regulated by testosterone (118).

Nonapeptides are important mediators of social behavior, such as aggression, reproduction, and paternal care, across vertebrates. Their effects are mediated by the presence of receptors and neuronal fibers found throughout the brain. AVP/AVT expression, in particular, has previously been examined across species, and it is canonically held that expression patterns in telencephalic regions of the brain are different between tetrapods and other vertebrates. Previous work has suggested that teleost fish only express AVT cell bodies within the POA-AH complex, and send projections to other telencephalic regions. However, here we find evidence for the presence of AVT preprohormone mRNA in regions previously not associated with AVT expression, such as the dorsomedial, ventral, and central regions of the A. burtoni telencephalon. Based on these results, it is worthwhile to reconsider the similarity in AVT/P expression patterns between teleosts and other vertebrates.

The original research reported here was performed under guidelines established and was reviewed and approved by the Institutional Animal Care and Use Committee at The University of Texas at Austin and in compliance with all local, state, and federal regulations.

CW, HH, and LH designed the studies; LH conducted the in situ hybridization study; CW and JN performed the qPCR experiments; LH, MR-S, and CW performed the data analysis; MR-S and HH wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.