Russ E. Carpenter

Serotonin decreases aggression via 5-HT1A receptors in the fighting fish Betta splendens

The role of the monoamine neurotransmitter serotonin (5-HT) in the modulation of conspecific aggression in the fighting fish (Betta splendens) was investigated using pharmacological manipulations. We used a fishs response to its mirror image as our index of aggressive behavior. We also investigated the effects of some manipulations on monoamine levels in the B. splendens brain. Acute treatment with 5-HT and with the 5-HT1A receptor agonist 8-OH-DPAT both decreased aggressive behavior; however, treatment with the 5-HT1A receptor antagonist WAY-100635 did not increase aggression. Chronic treatment with the selective serotonin reuptake inhibitor fluoxetine caused no significant changes in aggressive behavior and a significant decline in 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations. Treatment with the serotonin synthesis inhibitor p-chlorophenylalanine resulted in no change in aggression, yet serotonergic activity decreased significantly. Finally, a diet supplemented with L-tryptophan (Trp), the precursor to 5-HT, showed no consistent effects on aggressive behavior or brain monoamine concentrations. These results suggest a complex role for serotonin in the expression of aggression in teleost fishes, and that B. splendens may be a useful model organism in pharmacological and toxicological studies.

Parasite manipulation of brain monoamines in California killifish (Fundulus parvipinnis) by the trematode Euhaplorchis californiensis.

California killifish (Fundulus parvipinnis) infected with the brain-encysting trematode Euhaplorchis californiensis display conspicuous swimming behaviours rendering them more susceptible to predation by avian final hosts. Heavily infected killifish grow and reproduce normally, despite having thousands of cysts inside their braincases. This suggests that E. californiensis affects only specific locomotory behaviours. We hypothesised that changes in the serotonin and dopamine metabolism, essential for controlling locomotion and arousal may underlie this behaviour modification. We employed micropunch dissection and HPLC to analyse monoamine and monoamine metabolite concentrations in the brain regions of uninfected and experimentally infected fish. The parasites exerted density-dependent changes in monoaminergic activity distinct from those exhibited by fish subjected to stress. Specifically, E. californiensis inhibited a normally occurring, stress-induced elevation of serotonergic metabolism in the raphae nuclei. This effect was particularly evident in the experimentally infected fish, whose low-density infections were concentrated on the brainstem. Furthermore, high E. californiensis density was associated with increased dopaminergic activity in the hypothalamus and decreased serotonergic activity in the hippocampus. In conclusion, the altered monoaminergic metabolism may explain behavioural differences leading to increased predation of the infected killifish by their final host predators.

Corticotropin releasing factor induces anxiogenic locomotion in trout and alters serotonergic and dopaminergic activity

Corticotropin releasing factor (CRF) and serotonin (5-HT) are strongly linked to stress and anxiety in vertebrates. As a neuromodulator in the brain, CRF has anxiogenic properties often characterized by increased locomotion and stereotyped behavior in familiar environments. We hypothesized that expression of anxiogenic behavior in response to CRF will also be exhibited in a teleost fish. Rainbow trout were treated with intracerebroventricular (icv) injections of artificial cerebrospinal fluid (aCSF), 500 or 2000 ng ovine CRF, or not injected. Treatment with either dose of CRF elicited greater locomotion and pronounced head shaking behavior but did not influence water column position. Locomotor and head shaking behaviors may be analogous to the increased stereotypy evoked by icv CRF in rats and may reflect the expression of stress/anxiety behavior. Injection with either aCSF or CRF produced significant increases in plasma cortisol. The absence of behavioral changes in aCSF-injected fish suggests that the behavioral responses following CRF were not due to cortisol. Treatment with 2000 ng CRF significantly increased serotonin, 5-HIAA and dopamine concentrations in the subpallium and raphé and increased 5-HIAA in the preoptic hypothalamus (POA). Concurrent effects of CRF on central monoamines, locomotion and head shaking in trout suggest that anxiogenic properties of CRF are evolutionarily conserved. In addition, positive linear correlations between locomotion and serotonergic and dopaminergic function in the subpallium, POA and raphé nuclei suggest a locomotory function for these monoamines.

Characterization of cell proliferation throughout the brain of the African cichlid fish Astatotilapia burtoni and its regulation by social status.

New cells are added in the brains of all adult vertebrates, but fishes have some of the greatest potential for neurogenesis and gliogenesis among all taxa, partly due to their indeterminate growth. Little is known, however, about how social interactions influence cell proliferation in the brain of these fishes that comprise the largest group of vertebrates. We used 5‐bromo‐2′‐deoxyuridine (BrdU) to identify and localize proliferation zones in the telencephalon, diencephalon, mesencephalon, and rhombencephalon that were primarily associated with ventricular surfaces in the brain of the African cichlid fish Astatotilapia burtoni. Cell migration was evident in some regions by 1 day post injection, and many newborn cells coexpressed the neuronal marker HuC/D at 30 days, suggesting they had differentiated into neurons. To test the hypothesis that social status and perception of an opportunity to rise in rank influenced cell proliferation, we compared numbers of BrdU‐labeled cells in multiple brain nuclei among fish of different social status. Socially suppressed subordinate males had the lowest numbers of proliferating cells in all brain regions examined, but males that were given an opportunity to rise in status had higher cell proliferation rates within 1 day, suggesting rapid upregulation of brain mitotic activity associated with this social transition. Furthermore, socially isolated dominant males had similar numbers of BrdU‐labeled cells compared with dominant males that were housed in a socially rich environment, suggesting that isolation has little effect on proliferation and that reduced proliferation in subordinates is a result of the social subordination. These results suggest that A. burtoni will be a useful model to analyze the mechanisms of socially induced neurogenesis in vertebrates. J. Comp. Neurol. 520:3471–3491, 2012.

Corticotropin releasing factor influences aggression and monoamines: Modulation of attacks and retreats

Salmonids establish social hierarchies as a result of aggressive social interactions. The establishment of dominant or subordinate status is strongly linked to neuroendocrine responses mediated through the stress axis. In this study, we tested the effects of introcerebroventricular (icv) corticotropin releasing factor (CRF) on the behavioral outcome, plasma cortisol and monoamine function in trout subjected to a socially aggressive encounter. Rainbow trout were treated with an icv injection of artificial cerebrospinal fluid (aCSF), 500 or 2000 ng ovine CRF, or not injected. Fish were allowed to interact with a similarly sized conspecific for 15 min. Following the behavioral interaction, plasma cortisol and central monoamine concentrations were analyzed. Trout treated with CRF were victorious in approximately 66% of the aggressive encounters against aCSF-treated opponents. Trout injected with CRF exhibited a reduction in the total number of attacks and decreased latency to attack. When trout were divided into winners and losers, only victorious CRF-treated fish exhibited a reduced latency to attack and fewer retreats. Social stress increased cortisol levels in both winners and losers of aggressive interaction. This effect was enhanced with the additional stress incurred from icv injection of aCSF. However, icv CRF in addition to social stress decreased plasma cortisol in both winners and losers. While aggression stimulated significant changes in serotonergic and dopaminergic activity, the magnitude and direction were dependent on limbic brain region, CRF dose, and outcome of social aggression. With broad effects on aggressive behavior, anxiety, stress responsiveness, and central monoaminergic activity, CRF plays an important role in modulating the behavioral components of social interaction.

SEROTONIN, SOCIAL STATUS AND SEX CHANGE IN THE BLUEBANDED GOBY LYTHRYPNUS DALLI

In a variety of vertebrates, highly aggressive individuals tend to have high social status and low serotonergic function. In the sex changing fish Lythrypnus dalli, serotonin (5-HT) may be involved as a mediator between the social environment and the reproductive system because social status is a critical cue in regulating sex change. Subordination inhibits sex change in L. dalli, and it is associated with higher serotonergic activity in other species. We tested the hypothesis that high serotonergic activity has an inhibitory effect on sex change. In a social situation permissive to sex change, we administered to the dominant female implants containing the serotonin precursor 5-hydroxytryptophan (5-HTP). In a social situation not conducive to sex change, we administered either the serotonin synthesis inhibitor p-chlorophenylalanine (PCPA) or the 5-HT(1A) receptor antagonist p-MPPI. After three weeks we used HPLC to measure brain levels of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). We also performed PCPA, p-MPPI and fluoxetine injections in size-matched pairs of females to assess its effect on dominance status. Males and newly sex changed fish showed a trend for higher levels of 5-HIAA and 5-HT/5-HIAA ratio than females. The different implants treatments did not affect the probability of sex change. Interestingly, this species does not seem to fit the pattern seen in other vertebrates where dominant individuals have lower serotonergic activity than subordinates.

Modulation of monoamine neurotransmitters in fighting fish Betta splendens exposed to waterborne phytoestrogens

Endogenous estrogens are known to affect the activity of monoamine neurotransmitters in vertebrate animals, but the effects of exogenous estrogens on neurotransmitters are relatively poorly understood. We exposed sexually mature male fighting fish Betta splendens to environmentally relevant and pharmacological doses of three phytoestrogens that are potential endocrine disruptors in wild fish populations: genistein, equol, and β-sitosterol. We also exposed fish to two doses of the endogenous estrogen 17β-estradiol, which we selected as a positive control because phytoestrogens are putative estrogen mimics. Our results were variable, but the effects were generally modest. Genistein increased dopamine levels in the forebrains of B. splendens at both environmentally relevant and pharmacological doses. The environmentally relevant dose of equol increased dopamine levels in B. splendens forebrains, and the pharmacological dose decreased norepinephrine (forebrain), dopamine (hindbrain), and serotonin (forebrain) levels. The environmentally relevant dose of β-sitosterol decreased norepinephrine and dopamine in the forebrain and hindbrain, respectively. Our results suggest that sources of environmental phytoestrogens, such as runoff or effluent from agricultural fields, wood pulp mills, and sewage treatment plants, have the potential to modulate neurotransmitter activity in free-living fishes in a way that could interfere with normal behavioral processes.

Learning strategies during fear conditioning.

This paper describes a model of fear learning, in which subjects have an option of behavioral responses to impending social defeat. The model generates two types of learning: social avoidance and classical conditioning, dependent upon (1) escape from or (2) social subordination to an aggressor. We hypothesized that social stress provides the impetus as well as the necessary information to stimulate dichotomous goal-oriented learning. Specialized tanks were constructed to subject rainbow trout to a conditioning paradigm where the conditioned stimulus (CS) is cessation of tank water flow (water off) and the unconditioned stimulus (US) is social aggression from a larger conspecific. Following seven daily CS/US pairings, approximately half of the test fish learned to consistently escape the aggression to a neutral chamber through a small escape hole available only during the interaction. The learning curve for escaping fish was dramatic, with an 1100% improvement in escape time over 7 days. Fish that did not escape exhibited a 400% increase in plasma cortisol and altered brain monoamine response to presentation of the CS alone. Elevated plasma cortisol levels represent classical fear conditioning in non-escaping fish, while a lack of fear conditioning and a decreased latency to escape over the training period in escapers indicates learned escape.

Food deprivation explains effects of mouthbrooding on ovaries and steroid hormones, but not brain neuropeptide and receptor mRNAs, in an African cichlid fish

Feeding behavior and reproduction are coordinately regulated by the brain via neurotransmitters, circulating hormones, and neuropeptides. Reduced feeding allows animals to engage in other behaviors important for fitness, including mating and parental care. Some fishes cease feeding for weeks at a time in order to provide care to their young by brooding them inside the male or female parents mouth. Maternal mouthbrooding is known to impact circulating hormones and subsequent reproductive cycles, but neither the full effects of food deprivation nor the neural mechanisms are known. Here we ask what effects mouthbrooding has on several physiological processes including gonad and body mass, brain neuropeptide and receptor gene expression, and circulating steroid hormones in a mouthbrooding cichlid species, Astatotilapia burtoni. We ask whether any observed changes can be explained by food deprivation, and show that during mouthbrooding, ovary size and circulating levels of androgens and estrogens match those seen during food deprivation. Levels of gonadotropin-releasing hormone 1 (GnRH1) mRNA in the brain were low in food-deprived females compared to controls and in mouthbrooding females compared to gravid females. Levels of mRNA encoding two peptides involved in regulating feeding, hypocretin and cholecystokinin, were increased in the brains of food-deprived females. Brain mRNA levels of two receptors, GnRH receptor 2 and NPY receptor Y8c, were elevated in mouthbrooding females compared to the fed condition, but NPY receptor Y8b mRNA was differently regulated by mouthbrooding. These results suggest that many, but not all, of the characteristic physiological changes that occur during mouthbrooding are consequences of food deprivation.

Social status differences regulate the serotonergic system of a cichlid fish, Astatotilapia burtoni

Serotonin (5-HT) inhibits aggression and modulates aspects of sexual behaviour in many species, but the mechanisms responsible are not well understood. Here, we exploited the social dominance hierarchy of Astatotilapia burtoni to understand the role of the serotonergic system in long-term maintenance of social status. We identified three populations of 5-HT cells in dorsal and ventral periventricular pretectal nuclei (PPd, PPv), the nucleus of the paraventricular organ (PVO) and raphe. Dominant males had more 5-HT cells than subordinates in the raphe, but the size of these cells did not differ between social groups. Subordinates had higher serotonergic turnover in the raphe and preoptic area (POA), a nucleus essential for hypothalamic-pituitary–gonadal (HPG) axis function. The relative abundance of mRNAs for 5-HT receptor (5-HTR) subtypes 1A and 2A (htr1a, htr2a) was higher in subordinates, a difference restricted to the telencephalon. Because social status is tightly linked to reproductive capacity, we asked whether serotonin turnover and the expression of its receptors correlated with testes size and circulating levels of 11-ketotestosterone (11-KT). We found negative correlations between both raphe and POA serotonin turnover and testes size, as well as between htr1a mRNA levels and circulating 11-KT. Thus, increased serotonin turnover in non-aggressive males is restricted to specific brain nuclei and is associated with increased expression of 5-HTR subtypes 1A and 2A exclusively in the telencephalon.