Arginine vasotocin and androgen pathways are associated with mating system variation in North American cichlid fishes

Neuroendocrine pathways that regulate social behavior are remarkably conserved across divergent taxa. The neuropeptides arginine vasotocin/vasopressin (AVT/AVP) and their receptor V1a mediate aggression, space use, and mating behavior in male vertebrates. The hormone prolactin (PRL) also regulates social behavior across species, most notably paternal behavior. Both hormone systems may be involved in the evolution of monogamous mating systems. We compared AVT, AVT receptor V1a2, PRL, and PRL receptor PRLR1 gene expression in the brains as well as circulating androgen concentrations of free-living reproductively active males of two closely related North American cichlid species, the monogamous Herichthys cyanoguttatus and the polygynous Herichthys minckleyi. We found that H. cyanoguttatus males bond with a single female and together they cooperatively defend a small territory in which they reproduce. In H. minckleyi, a small number of large males defend large territories in which they mate with several females. Levels of V1a2 mRNA were higher in the hypothalamus of H. minckleyi, and PRLR1 expression was higher in the hypothalamus and telencephalon of H. minckleyi. 11-ketotestosterone levels were higher in H. minckleyi, while testosterone levels were higher in H. cyanoguttatus. Our results indicate that a highly active AVT/V1a2 circuit(s) in the brain is associated with space use and social dominance and that pair bonding is mediated either by a different, less active AVT/V1a2 circuit or by another neuroendocrine system.     

New insights into neuropeptide modulation of aggression: field studies of arginine vasotocin in a territorial tropical damselfish

The neuropeptides arginine vasotocin (AVT) and arginine vasopressin are key modulators of affiliation and aggression among non-mammalian and mammalian vertebrates, respectively. Here, we explored AVT's effect on aggression in a wild population of beaugregory damselfish, Stegastes leucostictus, a highly territorial species. Aggression by territorial males towards 'intruders' (bottled fishes) was assessed before and after each male received intramuscular injections of either AVT, Manning compound (an AVT V1a receptor antagonist), isotocin (the teleost homologue of mammalian oxytocin differing from AVT by two amino acids) or saline (vehicle control). Compared to saline controls, AVT and Manning increased and decreased aggression, respectively, while isotocin had no effect. Response selectivity was further established in a dose-response study that revealed an inverted U-shaped function. Compared to saline controls, aggression levels for low and high AVT doses were similar, while medium dose treatments were significantly greater. This type of behavioural response, the first that we know of for a vertebrate neuropeptide, could depend on the binding of AVT to both V1-type and other AVT or non-AVT receptors. The pattern revealed here for damselfish may be symptomatic of species- and context-dependent specificity of AVT's modulation of aggression across teleosts, as is currently proposed for tetrapods.     

Exogenous vasotocin alters aggression during agonistic exchanges in male Amargosa River pupfish (Cyprinodon nevadensis amargosae)

Pupfishes in the Death Valley region have rapidly differentiated in social behaviors since their isolation in a series of desert streams, springs, and marshes less than 20,000 years ago. These habitats can show dramatic fluctuations in ecological conditions, and pupfish must cope with the changes by plastic physiological and behavioral responses. Recently, we showed differences among some Death Valley populations in brain expression of arginine vasotocin (AVT). As AVT regulates both hydromineral balance and social behaviors in other taxa, these population differences may indicate adaptive changes in osmoregulatory and/or behavioral processes. To test whether AVT is relevant for behavioral shifts in these fish, here we examined how manipulations to the AVT system affect agonistic and reproductive behaviors in Amargosa River pupfish (Cyprinodon nevadensis amargosae). We administered exogenous AVT (0.1, 1, and 10 microg/g body weight) and an AVP V1 receptor antagonist (Manning compound, 2.5 microg/g body weight) intraperitoneally to males in mixed-sex groups in the laboratory. We found that AVT reduced the initiation of aggressive social interactions with other pupfish but had no effect on courtship. The effects of AVT were confirmed in males in the wild where AVT (1 microg/g body weight) reduced the aggressive initiation of social interactions and decreased aggressive responses to the behavior of other males. Combined, these results show that AVT can modulate agonistic behaviors in male pupfish and support the idea that variation in AVT activity may underlie differences in aggression among Death Valley populations.     

Manipulations of the AVT system shift social status and related courtship and aggressive behavior in the bluehead wrasse

Arginine vasotocin (AVT) and its mammalian homologoue arginine vasopressin (AVP) influence male sexual and aggressive behaviors in many species. We tested the effects of AVT and an AVP-V(1a) receptor antagonist on the display of alternative male tactics in a tropical coral reef fish, the bluehead wrasse Thalassoma bifasciatum. We gave AVT injections to territorial and nonterritorial males of the large and colorful phenotype (terminal phase) and an AVP-V(1a) receptor antagonist, Manning compound, to territorial males in the field. AVT increased courtship independent of status, while its effects on territoriality and aggression were dependent upon male status. In territorial males, AVT increased courtship and tended to decrease the number of chases toward initial phase individuals. In nonterritorial males, AVT increased courtship, chases toward initial phase individuals, and territorial behavior while decreasing feeding. These are all behaviors rarely seen in nonterritorial males, so AVT made these males act like territorial TP males. The AVP-V(1a) receptor antagonist had opposite effects. It decreased courtship and territorial defense, making these males act more like nonterritorial males. Manipulations of the AVT system shifted males within a single phenotype from the nonterritorial social status to the territorial social status and vice versa. Since the entire suite of behaviors related to territoriality was affected by AVT system manipulations, our results suggest that the AVT system may play a key role in motivation of behaviors related to mating.     

Involvement of arginine vasotocin in reproductive events in the male newt Cynops pyrrhogaster

Effects of arginine vasotocin (AVT) on reproductive events such as courtship behavior, pheromone release, and spermatophore discharge were investigated in the male newt Cynops pyrrhogaster. AVT enhanced the incidence and frequency of androgen-induced courtship behavior. In this case, AVT was likely to act centrally because the behavior was evoked with a much smaller amount of AVT when the hormone was administered intracerebroventricularly than when given intraperitoneally. Involvement of endogenous AVT in spontaneously occurring courtship behavior was also evidenced by the fact that administration of a V1 (vasopressor) receptor antagonist, [d(CH2)5(1), Tyr(Me)2, Arg8-vasopressin] suppressed the expression of the courtship behavior. The water in which AVT-treated males had been kept showed considerable female-attracting activity as compared with the water in which saline-injected males had been kept. Moreover, the content of sodefrin, a female-attracting pheromone in the abdominal gland, was decreased by the intraperitoneal injection of AVT, suggesting that the neurohypophyseal hormone stimulated the release of sodefrin from the abdominal gland into the water. AVT induced contraction of the excised abdominal gland concentration-dependently, and, again, the V1 receptor antagonist suppressed the AVT-induced contraction. Thus, we concluded that AVT induces the pheromone discharge, acting peripherally on a contractile structure of the abdominal gland. AVT was also found to induce spermatophore deposition in the male kept in the absence of the female. Administration of the V1 receptor blocker to the sexually developed males suppressed the spermatophore deposition. All these results indicate the involvement of AVT in reproductive events acting centrally and peripherally.     

Multiple mechanisms of phenotype development in the bluehead wrasse

Despite having detailed information on mechanisms mediating sex-typical behavior in many species, we have little understanding of whether the same mechanisms regulate these behaviors when they are performed in the same species under different social contexts. In the five field experiments of this study of bluehead wrasses (Thalassoma bifasciatum), a sex-changing fish, we examined the roles of arginine vasotocin (AVT) and the potent teleost androgen 11-ketotestosterone (11KT) in mediating sexual and aggressive behaviors typical of dominant males. We demonstrated that AVT appears necessary for the assumption of dominant territorial status in males and females, but is sufficient only in the socially dominant terminal phase (TP) male phenotype. Specifically, an AVP V(1) receptor antagonist prevented both TP males and females from gaining dominance over recently vacated territories. However, unlike TP males in a previous study, neither females nor initial phase males responded to AVT treatment with increases in display of TP male typical behaviors when under social conditions that inhibit sex change. Treating females with 11KT did not alter responsiveness to AVT, but did induce male coloration and courtship behavior that was not observed in oil-treated females. Combined with the results of a previous study, these results indicate that the ability of AVT to induce male-typical behavior differs among sexual phenotypes and that this differential responsiveness appears to be dependent on social context and not directly on exposure to 11KT. Furthermore, since 11KT can induce courtship behavior in females that is not affected by AVT, there may be different hormonal mechanisms mediating courtship behavior under different social contexts.     

Population divergence in plasticity of the AVT system and its association with aggressive behaviors in a Death Valley pupfish

Behavioral differences can evolve rapidly in allopatry, but little is known about the neural bases of such changes. Allopatric populations of Amargosa pupfish (Cyprinodon nevadensis) vary in aggression and courtship behaviors in the wild. Two of these wild populations were recently found to differ in brain expression of arginine vasotocin (AVT)--a peptide hormone shown previously to modulate aggression in pupfish. These populations have been isolated for less than 4000 years, so it remained unclear whether the differences in behavior and neural AVT phenotype were evolved changes or plastic responses to ecologically dissimilar habitats. Here, I tested whether these population differences have a genetic basis by examining how aggressive behavior and neural AVT phenotype responded to ecologically relevant variation in salinity (0.4 ppt or 3 ppt) and temperature (stable or daily fluctuating). Pupfish from Big Spring were more aggressive than pupfish from the Amargosa River when bred and reared under common laboratory conditions. Morphometric analysis of preoptic AVT immunoreactivity showed that the populations differed in how the AVT system responded to salinity and temperature conditions, and revealed that this plasticity differed between parvocellular and magnocellular AVT neuron groups. Both populations also showed relationships between neural AVT phenotype and aggression in the rearing environment, although populations differed in how aggression related to variation in magnocellular AVT neuron size. Together, these results demonstrate that the pupfish populations have diverged in how physical and social conditions affect the AVT system, and provide evidence that the AVT system can evolve quickly to ecologically dissimilar environments.     

Central Vasopressin Administration Regulates the Onset of Facultative Paternal Behavior in Microtus pennsylvanicus (Meadow Voles)

Pharmacological experiments have implicated a role for central arginine vasopressin (AVP) in regulating paternal behavior in monogamous prairie voles. Although nonmonogamous meadow voles exhibit appreciable paternal care when housed under winter, short day lengths (SD), no research has examined whether the same neurobiological systems are involved in regulating paternal behavior in a nonmonogamous species when it behaves paternally. The goal of these experiments was to determine whether central administration of AVP, but not cerebrospinal fluid (CSF), affected the suppression of pup-directed aggression and/or the onset of paternal behavior in meadow voles. Data from experiment 1 implicated a role for AVP in facilitating changes in male behavior: central administration of 1 ng of AVP (but not 3 ng or CSF) inhibited pup-directed aggression in previously pup-aggressive males, and 3 ng of AVP (but not 1 ng or CSF) induced paternal behavior in previously nonpaternal males. In contrast, AVP (1 and 3 ng) did not enhance paternal behavior in already paternal males. Experiment 2 tested the specificity of AVP. Previous research indicated that 24 h of unmated cohabitation with a female reliably induced paternal behavior in SD males. Hence, experiment 2 examined whether administration of a V_1a AVP antagonist (AVPA), but not CSF, prior to 24 h of unmated cohabitation would block the onset of paternal behavior. Males that received CSF displayed paternal behavior faster and engaged in more investigatory and paternal behaviors than males that received AVPA. Thus, pharmacological experiments support the hypothesis that AVP likely regulates paternal behavior in both facultatively and consistently paternal vole species.     

Parasites, aggression and dominance in male upland bullies

The effects of the digenean trematode parasite Telogaster opisthorchis on aggression and competitive ability in male upland bullies ( Gobiomorphus breviceps ) were examined using mirror-image stimulation (MIS) techniques and dyadic contests for nest sites between pairs of males. Parasite load had no significant effect on male aggression or success in dyadic contests, nor was aggression a predictor of the likely winner of these contests. The results are discussed with relation to the role of parasites in male-male competition and to possible problems in using MIS techniques for studies on dominance and aggression in fish.     

The regulation of social recognition, social communication and aggression: vasopressin in the social behavior neural network

Neuropeptides in the arginine vasotocin/arginine vasopressin (AVT/AVP) family play a major role in the regulation of social behavior by their actions in the brain. In mammals, AVP is found within a circuit of recriprocally connected limbic structures that form the social behavior neural network. This review examines the role played by AVP within this network in controlling social processes that are critical for the formation and maintenance of social relationships: social recognition, social communication and aggression. Studies in a number of mammalian species indicate that AVP and AVP V1a receptors are ideally suited to regulate the expression of social processes because of their plasticity in response to factors that influence social behavior. The pattern of AVP innervation and V1a receptors across the social behavior neural network may determine the potential range and intensity of social responses that individuals display in different social situations. Although fundamental information on how social behavior is wired in the brain is still lacking, it is clear that different social behaviors can be influenced by the actions of AVP in the same region of the network and that AVP can act within multiple regions of this network to regulate the expression of individual social behaviors. The existing data suggest that AVP can influence social behavior by modulating the interpretation of sensory information, by influencing decision making and by triggering complex motor outputs. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.     

Arginine vasotocin induces sexual behavior of newts by acting on cells in the brain

To investigate whether arginine vasotocin (AVT) acts on target cells in the brain of Taricha granulosa (a urodele amphibian), the behavioral effects of intracerebroventricular (ICV) and intraperitoneal (IP) injections of AVT were compared. Male newts exhibited the greatest sexual activity (amplectic clasping) following an ICV injection of 0.1 μg AVT. Another study showed that nanogram quantities of AVT, administered ICV, stimulated the behavior. An ICV injection of an antagonist to arginine vasopressin, d(CH₂)₅Tyr(Me)AVP, or an anti-AVT immune serum significantly inhibited the sexual behavior. Intracranial implants of 17β-estradiol (E₂) or 5α-dihydrotestosterone (DHT) in castrated males maintained the behavioral response to an injection of AVT. Another study found that an IP injection of DHT or E₂ did not increase the incidence of newt sexual behavior during the 8 hours following the injection.     

Central vasopressin administration regulates the onset of facultative paternal behavior in microtus pennsylvanicus (meadow voles)

Pharmacological experiments have implicated a role for central arginine vasopressin (AVP) in regulating paternal behavior in monogamous prairie voles. Although nonmonogamous meadow voles exhibit appreciable paternal care when housed under winter, short day lengths (SD), no research has examined whether the same neurobiological systems are involved in regulating paternal behavior in a nonmonogamous species when it behaves paternally. The goal of these experiments was to determine whether central administration of AVP, but not cerebrospinal fluid (CSF), affected the suppression of pup-directed aggression and/or the onset of paternal behavior in meadow voles. Data from experiment 1 implicated a role for AVP in facilitating changes in male behavior: central administration of 1 ng of AVP (but not 3 ng or CSF) inhibited pup-directed aggression in previously pup-aggressive males, and 3 ng of AVP (but not 1 ng or CSF) induced paternal behavior in previously nonpaternal males. In contrast, AVP (1 and 3 ng) did not enhance paternal behavior in already paternal males. Experiment 2 tested the specificity of AVP. Previous research indicated that 24 h of unmated cohabitation with a female reliably induced paternal behavior in SD males. Hence, experiment 2 examined whether administration of a V(1a) AVP antagonist (AVPA), but not CSF, prior to 24 h of unmated cohabitation would block the onset of paternal behavior. Males that received CSF displayed paternal behavior faster and engaged in more investigatory and paternal behaviors than males that received AVPA. Thus, pharmacological experiments support the hypothesis that AVP likely regulates paternal behavior in both facultatively and consistently paternal vole species.     

Developmental effects of vasotocin and nonapeptide receptors on early social attachment and affiliative behavior in the zebra finch

Zebra finches demonstrate selective affiliation between juvenile offspring and parents, which, like affiliation between pair partners, is characterized by proximity, vocal communication and contact behaviors. This experiment tested the hypothesis that the nonapeptide arginine vasotocin (AVT, avian homologue of vasopressin) and nonapeptide receptors play a role prior to fledging in the development of affiliative behavior. Zebra finch hatchlings of both sexes received daily intracranial injections (post-hatch days 2–8) of either AVT, Manning Compound (MC, a potent V1a receptor antagonist) or saline (vehicle control). The social development of both sexes was assessed by measuring responsiveness to isolation from the family and subsequent reunion with the male parent after fledging. In addition, we assessed the changes in affiliation with the parents, unfamiliar males, and unfamiliar females each week throughout juvenile development. Compared to controls, MC subjects showed decreased attachment to the parents and MC males did not show the normal increase in affiliative interest in opposite sex individuals as they reached reproductive maturity. In contrast, AVT subjects showed a sustained affiliative interest in parents throughout development, and males showed increased interest in opposite sex conspecifics as they matured. These results provide the first evidence suggesting that AVT and nonapeptide receptors play organizational roles in social development in a bird.     

Integrating resource defence theory with a neural nonapeptide pathway to explain territory-based mating systems

The ultimate-level factors that drive the evolution of mating systems have been well studied, but an evolutionarily conserved neural mechanism involved in shaping behaviour and social organization across species has remained elusive. Here, we review studies that have investigated the role of neural arginine vasopressin (AVP), vasotocin (AVT), and their receptor V1a in mediating variation in territorial behaviour. First, we discuss how aggression and territoriality are a function of population density in an inverted-U relationship according to resource defence theory, and how territoriality influences some mating systems. Next, we find that neural AVP, AVT, and V1a expression, especially in one particular neural circuit involving the lateral septum of the forebrain, are associated with territorial behaviour in males of diverse species, most likely due to their role in enhancing social cognition. Then we review studies that examined multiple species and find that neural AVP, AVT, and V1a expression is associated with territory size in mammals and fishes. Because territoriality plays an important role in shaping mating systems in many species, we present the idea that neural AVP, AVT, and V1a expression that is selected to mediate territory size may also influence the evolution of different mating systems. Future research that interprets proximate-level neuro-molecular mechanisms in the context of ultimate-level ecological theory may provide deep insight into the brain-behaviour relationships that underlie the diversity of social organization and mating systems seen across the animal kingdom.     

Brain levels of arginine-vasotocin and isotocin in dominant and subordinate males of a cichlid fish

The nonapeptides arginine-vasotocin (AVT) and isotocin (IT), which are the teleost homologues of arginine-vasopressin and oxytocin in mammals, have well established peripheral effects on osmoregulation and stress response, and central effects on social behavior. However, all studies that have looked so far into the relationship between these nonapeptides and social behavior have used indirect measures of AVT/IT activity (i.e. immunohistochemistry of AVT/IT immunoreactive neurons, or AVT/IT or their receptors mRNA expression with in situ hybridization or qPCR) and therefore direct measures of peptide levels in relation to social behavior are still lacking. Here we use a recently developed high-performance liquid chromatography analysis with fluorescence detection (HPLC-FL) method to quantify the levels of both AVT and IT in macro-dissected brain areas [i.e. olfactory bulbs, telencephalon, diencephalon, optic tectum, cerebellum, and hindbrain (= rhombencephalon minus cerebellum)] and pituitary of dominant and subordinate male cichlid fish (Oreochromis mossambicus). The pituitary shows higher levels of both peptides than any of the brain macroareas, and the olfactory bulbs have the highest AVT among all brain areas. Except for IT in the telencephalon there is a lack of correlations between central levels and pituitary peptide levels, suggesting an independent control of hypophysial and CNS nonapeptide secretion. There were also no correlations between AVT and IT levels either for each brain region or for the pituitary gland, suggesting a decoupled activity of the AVT and IT systems at the CNS level. Subordinate AVT pituitary levels are significantly higher than those of dominants, and dominant hindbrain IT levels are significantly higher than those of subordinates, suggesting a potential involvement of AVT in social stress in subordinate fish and of IT in the regulation of dominant behavior at the level of the hindbrain. Since in this species dominant males use urine to communicate social status and since AVT is known to have an antidiuretic effect, we have also investigated the effect of social status on urine storage. As predicted, dominant males stored significantly more urine than subordinates. Given these results we suggest that AVT/IT play a key role in orchestrating social phenotypes, acting both as central neuromodulators that promote behavioral plasticity and as peripheral hormones that promote integrated physiological changes.     

Paternal behavior increases testosterone levels in offspring of the California mouse

Paternal care during early development influences pup survivorship in the monogamous and biparental California mouse, Peromyscus californicus. Moreover, paternal pup retrievals impact development of adult offspring aggression and the neuropeptide vasopressin, yet little is known about the underlying mechanisms of these developmental changes. Because testosterone can increase arginine vasopressin and aggression, we hypothesized that paternal pup retrievals increase testosterone levels in prepubertal male P. californicus pups. Male pups were assigned to one of three groups: hormonal baseline, nonretrieval control, or retrieval. On postnatal days 18-21, all pups and the mother were removed from the cage, and the focal male pup was placed either outside of the nest to elicit paternal retrievals (retrieval group) or in the nest to discourage paternal retrievals (nonretrieval group). Testosterone was elevated at 45-min, but not 90-min, post-manipulation in retrieved compared to nonretrieved pups. Moreover, there was a significant positive correlation between pup retrievals and testosterone in the 45-min group. This rapid testosterone rise in response to paternal retrievals may facilitate an increase in aggression and vasopressin in adult offspring. Therefore, this period of development previously viewed as hormonally quiescent may be more active in response to paternal behavior than previously thought.     

Nonapeptides and social behavior in fishes

The nonapeptide hormones arginine vasotocin and isotocin play important roles in mediating social behaviors in fishes. Studies in a diverse range of species demonstrate variation in vasotocin neuronal phenotypes across within and between sexes and species as well as effects of hormone administration on aggressive and sexual behaviors. However, patterns vary considerably across species and a general explanatory model for the role of vasotocin in teleost sociosexual behaviors has proven elusive. We review these findings, examine potential explanations for the lack of agreement across studies, and propose a model based on the parvocellular AVT neurons primarily mediating social approach and subordinance functions while the magnocellular and gigantocellular AVT neurons mediate courtship and aggressive behaviors. Isotocin neuronal phenotypes and effects on behavior are relatively unstudied, but research to date suggests this will be a fruitful line of inquiry. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.     

Effect of vasotocin on locomotor activity in bullfrogs varies with developmental stage and sex

Although neurohypophysial peptides are present in many regions of the developing and adult bullfrog (Rana catesbeiana) brain, the function of these peptides remains unclear. To investigate possible behavioral actions, we examined locomotor activity following peptide injection in bullfrogs at various developmental stages. An intraperitoneal (ip) injection of arginine vasotocin (AVT) in tadpoles (stages V, X, or XVII) produced an immediate and dose-dependent inhibition of locomotor activity. On the other hand, AVT stimulated activity when administered ip to juvenile or adult female bullfrogs, but did not influence activity in juvenile or adult males. The minimum effective dose of AVT, when injected directly into the brain of tadpoles, was 100-fold less than that observed when injected ip, suggesting a central nervous system site of action for this peptide. A vasopressin receptor antagonist (d(CH2)5[Tyr(Me)2]AVP administered ip or icv) significantly increased locomotor activity in tadpoles, compared to controls. Oxytocin, vasopressin, and AVP4-9 inhibited activity in tadpoles while mesotocin, des Gly(NH2)AVP, and pressinoic acid had no significant effect. Injection of PGF2 alpha also significantly decreased activity levels in tadpoles. However, pretreatment of tadpoles with indomethacin, a prostaglandin synthesis inhibitor, did not prevent the behavioral effects of AVT, suggesting that prostaglandin synthesis is not required for this response. In summary, AVT influenced locomotor activity in bullfrog tadpoles and female frogs. This effect shifted during development from an inhibitory action in tadpoles to a stimulatory effect in metamorphosed female frogs. The effect of AVT on juvenile and adult frog locomotion was sexually dimorphic, as this peptide altered female behavior but not male behavior.     

Neonatal buspirone modulates the intermale aggression in adult mice

Mice of two strains with different levels of male aggression (RSB and RLB) were subjected to daily injections of 5-HT1A receptor agonist buspirone (25 microg) on the 2nd - 6th postnatal days. This neonatal treatment augmented the aggressive behavior (tested in the dyadic contests with non-aggressive A/Sn males) in aggressive RSB mice and reduced aggression in less aggressive RLB. Correlations with different signs were found between the 5-HT and 5-HIAA levels in the neocortex, hippocampus, and hypothalamus and behavioral indices of aggression in RSB and RLB males. The remote effects of neonatal buspirone in these two mice strains presumably depend on genotype-related features of ontogeny of the 5-HT system.