Effects of serotonin and serotonin analogs on posture and agonistic behavior in crayfish

Exogenous serotonin has been shown to induce an elevated, flexed posture in crustaceans and has also been hypothesized to enhance aggressive behavior. We conducted three experiments to further investigate the effects of serotonin and serotonin analogs on posture and agonistic behavior in the crayfish Procambarus clarkii. In the first experiment, we recorded behavioral responses to five different concentrations of serotonin injected into the ventral hemolymph sinus. The amine elicited a series of behaviors including the characteristic high, flexed posture, but none were clearly associated with aggression. In our second experiment, we tested serotonin and four serotonin receptor agonists [1-(3-chlorophenyl)piperazine dihydrochloride, 2-methyl-5-hydroxytryptamine maleate, 5-carboxamidotryptamine maleate and alpha-methyl-5-hydroxytryptamine maleate] and measured the ability of each agonist to mimic the actions of the amine. High concentrations of 1-(3-chlorophenyl)piperazine dihydrochloride most closely mimicked the actions of serotonin; 5-carboxamidotryptamine maleate induced a high stance, but did not otherwise induce effects similar to serotonin. In our third experiment, we conducted an analysis of fighting behavior between pairs of crayfish that had received injections of control saline, serotonin, or 5-carboxamidotryptamine maleate. Serotonin generally reduced the level of aggression between opponents, whereas 5-carboxamidotryptamine maleate enhanced the performance of several agonistic behaviors.     

Chronic alterations in serotonin function: dynamic neurochemical properties in agonistic behavior of the crayfish, Orconectes rusticus

The biogenic amine serotonin [5-hydroxytryptamine (5-HT)] has received considerable attention for its role in behavioral phenomena throughout a broad range of invertebrate and vertebrate taxa. Acute 5-HT infusion decreases the likelihood of crayfish to retreat from dominant opponents. The present study reports the biochemical and behavioral effects resulting from chronic treatment with 5-HT-modifying compounds delivered for up to 5 weeks via silastic tube implants. High performance liquid chromatography with electrochemical detection (HPLC-ED) confirmed that 5,7-dihydroxytryptamine (5,7-DHT) effectively reduced 5-HT in all central nervous system (CNS) areas, except brain, while a concurrent accumulation of the compound was observed in all tissues analyzed. Unexpectedly, two different rates of chronic 5-HT treatment did not increase levels of the amine in the CNS. Behaviorally, 5,7-DHT treated crayfish exhibited no significant differences in measures of aggression. Although treatment with 5-HT did not elevate 5-HT content in the CNS, infusion at a slow rate caused animals to escalate more quickly while 5-HT treatment at a faster rate resulted in slower escalation. 5,7-DHT is commonly used in behavioral pharmacology and the present findings suggest its biochemical properties should be more thoroughly examined. Moreover, the apparent presence of powerful compensatory mechanisms indicates our need to adopt an increasingly dynamic view of the serotonergic bases of behavior like crayfish aggression.     

Amines and motivated behaviors: a simpler systems approach to complex behavioral phenomena

Recent investigations in invertebrate neurobiology have opened up new lines of research into the basic roles of behavioral, neurochemical, and physiological effects in complex behavioral phenomena, such as aggression and drug-sensitive reward. This review summarizes a body of quantitative work, which identifies biogenic amines as a pharmacological substrate for motivated behaviors in the crayfish, Orconectes rusticus. Specifically, this paper details progress that has (1) explored links between serotonin and an individuals aggressive state, and (2) demonstrated the existence of crayfish reward systems that are sensitive to human drugs of abuse, such as psychostimulants. First, we summarize a set of experimental approaches that explore aggression in crayfish and the significance of aminergic systems in its control. Agonistic behavior in crustaceans can be characterized within a quantitative framework; different types of behavioral plasticity in aggressive behavior are in need of physiological explanation, and pharmacological intervention involving serotonergic systems bring about characteristic changes in behavior. A second set of experiments demonstrates that psychostimulants (cocaine and D-amphetamine) serve as rewards when an intra-circulatory infusion is coupled to a distinct visual environment. Work in novel model systems such as crayfish constitutes a useful comparative approach to the study of aggression and drug addiction.     

Fish on Prozac: a simple, noninvasive physiology laboratory investigating the mechanisms of aggressive behavior in Betta splendens

The neuromodulator serotonin is an important regulator of aggressive behavior in vertebrates. Experimentally increasing synaptic levels of serotonin with fluoxetine, a selective serotonin reuptake inhibitor, has been shown to reliably decrease the expression of aggressive behavior. Here, we describe a method by which fluoxetine can be noninvasively administered to male Betta splendens (an attractive model for the study of aggressive behavior) and describe a simple laboratory exercise that allows students to experimentally investigate the physiological mechanisms of aggressive behavior. We demonstrate that relatively short-term exposure (3 h) of male bettas to as little as 3 microg/ml of fluoxetine-treated aquarium water is sufficient to reduce the expression of specific aggressive behaviors. We emphasize the physiological concepts that can be addressed with this exercise, including the role of the serotonergic system in regulating aggression, and the interplay of environmental contaminants and physiology in regulating the expression of behavior. We also highlight important aspects of experimental design. This exercise can be flexibly altered to accommodate one or several laboratory periods. It is also low cost, is low impact to the animals, and requires minimal preparation time for instructors.     

Serotonin, social status and aggression

Serotonin, social status and aggression appear to be linked in many animal species, including humans. The linkages are complex,and, for the most part, details relating the amine to the behavior remain obscure. During the past year, important advances have been made in a crustacean model system relating serotonin and aggression. The findings include the demonstration that serotonin injections will cause transient reversals in the unwillingness of subordinate animals to engage in agonistic encounters, and that at specific synaptic sites involved in activation of escape behavior, the direction of the modulation by serotonin depends on the social status of the animal.     

Serotonergic modulation of footshock induced aggression in paired rats.

Footshock induced aggression (FIA) was induced in paired rats and three paradigms of aggressive behaviour were recorded, namely, latency to fight (LF), total period of physical contact (TPP) and cumulative aggression scores (CAS). The effects of increasing or decreasing central serotonergic activity, by using a number of pharmacological agents with well defined effects on rat brain serotonin, were investigated on FIA and on FIA augmented by apomorphine, a dopamine receptor agonist. The results show that centrally administered serotonin, the serotonin precursor, 5-hydroxytryptophan administered with clorgyline, a selective MAO A inhibitor, quipazine, a serotonin receptor agonist, and fluoxetine, a selective inhibitor of neuronal re-uptake of serotonin, attenuated all paradigms of FIA and apomorphine induced potentiation of FIA. On the contrary, the other re-uptake inhibitor used, citalopram, appeared to have a dual effect and decreased LF and CAS, while increasing TPP. The serotonin synthesis inhibitor, p-chlorophenylalanine and the selective serotonin receptor (5-HT2) antagonist, ketanserin, augmented all paradigms of FIA per se and apomorphine induced augmentation of FIA. However, the other serotonin receptor antagonist used, metergoline, which blocks both 5-HT1 and 5-HT2 receptor subtypes, attenuated FIA per se but decreased only CAS in apomorphine induced increase in FIA. The data confirm the inhibitory effect of the central serotonergic system on aggressive behaviour and the inverse relationship existing between it and the central dopaminergic system in the modulation of FIA, as has also been confirmed in earlier biochemical investigations from this laboratory. The data has been discussed in the light of existing knowledge on serotonin receptor subtypes and the presence of modulatory serotonergic heteroreceptors on central dopaminergic neurones.     

Agonistic behavior in naïve juvenile lobsters depleted of serotonin by 5,7-dihydroxytryptamine

We have been exploring the role of serotonin in fighting behavior in lobsters using a specific model of agonistic behavior, the establishment of hierarchical relationships between pairs of socially naive juvenile lobsters. We selected this model because the behavior is easily evoked, readily quantifiable, and the effects of experience are eleminated by using socially naive animals. In these studies we injected a specific neurotoxin, 5,7-dihydroxytryptamine, into juvenile lobsters over a 4-week period and then measured the effects on fighting behavior. This treatment reduces the levels of serotonin in the nervous system and immunocytochemical studies show a dramatic reduction in neuropil staining for the amine. Control animals received vehicle injection alone. All injected animals were paired against larger or smaller non-injected opponents, and three successive 30-min fights were carried out and statistically analyzed. The results were surprising: As with elevations of serotonin, reduced levels of serotonin increased the amount of time animals engaged in fighting behavior. No significant effects were seen on who initiated encounters, who retreated first, or who the eventual winner would be. Thus, in this model, elevation or reduction of serotonergic function increases the tendency of animals to engage in agonistic encounters.     

Fluoxetine Partly Exerts its Actions Through GABA: A Neurochemical Evidence

Fluoxetine, as a serotonin re-uptake inhibitor augments serotonin concentration within the synapse by inhibiting the serotonin transporter. The contribution of amino acids has also been shown in depression. We hypothesized that fluoxetine exerts its actions at least in part by intervening brain signaling operated by amino acid transmitters. Therefore the aim of this study is to supply neurochemical evidence that fluoxetine produces changes in amino acids in cerebrospinal fluid of rats. Sprague-Dawley rats were anesthetized and concentric microdialysis probes were implanted stereotaxically into the right lateral ventricle. Intraperitoneal fluoxetine (2.5 or 5 mg/kg) or physiological saline was administered and the probes were perfused with artificial cerebrospinal fluid at a rate of 1 μl/min. In the chronic fluoxetine group, the rats were treated daily with oral fluoxetine solution or inert syrup for 3 weeks. The microdialysis probes were placed on the 21st day and perfused the next day. Fluoxetine was ineffective in changing the cerebrospinal fluid GABA levels at the dose of 2.5 mg/kg but produced a significant increase in the perfusates following injection of 5 mg/kg of fluoxetine (P < 0.05). Oral fluoxetine administration (5 mg/kg) for 21 days also elevated the CSF GABA levels by approximately 2-fold (P < 0.05). l-glutamic acid levels were not affected in all groups. These neurochemical findings show that fluoxetine, a selective serotonin re-uptake inhibitor affects brain GABA levels indirectly, and our results suggest that acute or chronic effects may be involved in beneficial and/or adverse effects of the drug.     

Effects of fluoxetine on growth and behavior in the crayfish Orconectes rusticus

This laboratory study examined the effects of the specific serotonin reuptake inhibitor fluoxetine on growth following molting and on a range of behaviors in the crayfish Orconectes rusticus. For growth experiments, male Form I and Form II crayfish were weighed and measured and placed individually in water containing 0–500 μg/L of fluoxetine. They were held in fluoxetine or control water until they molted and were reweighed two weeks post-molt. In behavior experiments, juvenile and adult animals were held individually in 0, 2, 200, or 500 μg/L of fluoxetine for 10 days and tested in an open field arena to assess locomotion, thigmotaxis, sheltering, and habituation to a novel environment. Under our laboratory conditions, crayfish exposed to fluoxetine at 500 μg/L showed significantly enhanced growth: post-molt Form I animals had greater body weight and post-molt Form II animals had greater carapace length, relative to controls. In open field tests, juvenile crayfish exposed to 2 and 500 μg/L fluoxetine displayed significantly reduced locomotion compared to controls. The results indicate that crayfish growth and locomotion can be manipulated by short-term exposure to ambient fluoxetine, suggesting that this means of exposure may offer a useful and noninvasive way to examine drug effects in freely moving animals. However, effects were only observed at concentrations well above fluoxetine levels currently reported in the environment. This suggests that O. rusticus may be relatively resistant to this form of pharmaceutical pollution but whether effects would occur following long-term exposure to lower concentrations is unknown.     

Serotonergic effects on feeding, but not hypothalamus-pituitary-adrenal secretion, are altered in ovine pregnancy

In ovine pregnancy, as in human pregnancy, hypothalamus-pituitary-adrenal activity is chronically increased. These studies were designed to test the hypotheses that expression of serotonergic genes and responsiveness to serotonin are increased in pregnancy. We tested the stimulatory effect of an acute, intracerebroventricular injection of the serotonin reuptake inhibitor fluoxetine on plasma ACTH and cortisol in ewes during late pregnancy or postpartum. We also tested the effect of lower-dose, longer-term stimulation by intracerebroventricular infusion of fluoxetine in pregnant and nonpregnant ewes over 6 days. Overall, we found that the stimulatory effect of fluoxetine on ACTH and cortisol was not significantly different between late-gestation and nonpregnant ewes, although the effect of acute fluoxetine administration was inversely related to plasma progesterone concentrations. Also, there were no differences in hypothalamic expression of the glucocorticoid and mineralocorticoid receptors, corticotropin-releasing hormone, AVP, the serotonin reuptake transporter, or the serotonin [5-hydroxytryptamine (5-HT)] receptors 5-HT(1A) and 5-HT(2A) with pregnancy or fluoxetine treatment. However, chronic fluoxetine infusion reduced food intake in the nonpregnant, but not pregnant, ewes. Expression of proopiomelanocortin mRNA in the hypothalamus was reduced in pregnant compared with nonpregnant ewes. Our results indicate that pregnancy does not increase responsiveness of ACTH and cortisol to serotonergic stimulation but, rather, that progesterone reduces the ACTH response. In addition, we found a reduced ability of serotonin to inhibit feeding in the pregnant ewes, consistent with a reduction in anorexic mechanisms in the pregnant state.     

Behavioral effects of serotonin and serotonin agonists in two crayfish species, Procambarus clarkii and Orconectes rusticus

Exogenous serotonin elicits several behaviors in Procambarus clarkii, including a flexed, elevated posture, reduced locomotion, and changes in aggressive behavior. We conducted experiments to determine if several serotonin agonists mimicked the behavioral effects of serotonin in two crayfish species, P. clarkii and Orconectes rusticus. Drugs tested were 1-(3-Chlorophenyl)-piperazine dihydrochloride (mCPP), Oxymetazoline, 2,5-dimethoxy-4-iodoamphetamine (DOI), CGS-12066A, and (+/-)-8-hydroxy-2-(di-n-dipropylamino) tetralin (8-OH-DPAT). In P. clarkii, mCPP most closely mimicked the effects of serotonin, significantly increasing the performance of the flexed, elevated posture and reducing locomotion; 8-OH-DPAT significantly reduced locomotion as well. Both of these drugs produced significant increases in elevated posture and decreases in locomotion in O. rusticus, and in this species, the drugs at test concentrations were more effective in eliciting these effects than serotonin. The effects of the drugs on behaviors performed during fighting bouts were variable. In both species, only 8-OH-DPAT significantly reduced several agonistic behaviors, and no agonist or 5-HT itself produced significant increases in agonistic behavior.     

Ritualized submission and the reduction of aggression in an invertebrate

Ritualized behaviors that signify acceptance of a dominance relationship and reduce aggression between rivals are a common feature of vertebrate social behavior. Although some invertebrates, including crayfish, lobsters, and ants, display dominance postures, more complex dominance rituals and their effects on fitness have not been reported. We found that crayfish display such a complex ritual, when two males engaged in pseudocopulatory behavior to signify their dominance relationship. This was followed by a reduction in aggression and an increased likelihood of the subordinate's survival. Pseudocopulation was initiated by the eventual dominant and could be accepted or refused by the eventual subordinate. The frequency of aggressive behavior declined significantly during the first hour in all pairs that pseudocopulated but remained high in pairs that did not. Whereas all the subordinate members of pairs that pseudocopulated survived the initial 24 hr of pairing, half of subordinates that did not pseudocopulate were killed during that time. This differential mortality indicates that the reduction of aggression induced by the pseudocopulatory ritual directly enhances the differential survival of male crayfish that engage in this behavior.     

Role of the olfactory pathway in agonistic behavior of crayfish, <Emphasis Type="Italic">Procambarus clarkii</Emphasis>

Crayfish establish social dominance hierarchies through agonistic interactions, and these hierarchies are maintained through assessment of social status. Chemical signals influence several aspects of fighting behavior, but the specific chemosensory sensilla involved in detecting these signals in crayfish are unknown. The goal of our study was to examine the importance of aesthetasc sensilla—olfactory sensors on the antennules of decapod crustaceans—in regulating changes in fighting behavior in crayfish, Procambarus clarkii, over the course of repeated pairings. We selectively ablated aesthetascs from pairs of crayfish after the first day of trials and compared the behavior of these ablated animals to that of pairs of intact controls. Results show that unablated crayfish significantly decreased the number and duration of fights over repeated pairings, whereas crayfish lacking aesthetascs continued to engage in similar amounts of fighting across all three trial days. This difference shows that aesthetascs regulate fighting behavior in P. clarkii.     

Evidence that serotonin reuptake modulators increase the density of serotonin innervation in the forebrain

The mechanism of action of commonly used antidepressants remains an issue of debate. In the experiments reported here we studied the effects of three representative compounds, the selective serotonin reuptake inhibitor fluoxetine, the selective serotonin reuptake enhancer tianeptine and the selective norepinephrine reuptake inhibitor desipramine on the structure of central serotonin pathways after a 4-week administration. We found that the serotonin modulators fluoxetine and tianeptine, but not desipramine, increase the density of 5-HT and serotonin transporter (SERT)-immunoreactive axons in the neocortical layer IV and certain forebrain limbic areas, such as piriform cortex and the shell region of nucleus accumbens. These changes were noted in the absence of a significant effect of serotonin antidepressants on the expression of tryptophan hydroxylase (TPH-2), i.e. the rate-limiting enzyme for 5-HT biosynthesis and of SERT at the mRNA level. In addition, we found that anterogradely filled terminal axons from injections of biotinylated dextran amine into the dorsal raphe showed significantly more branching in animals treated with fluoxetine compared with animals treated with liposyn vehicle. Our findings suggest that antidepressants may exert very selective structural effects on their cognate monoamine systems in normal animals and raise the possibility that neurotrophic mechanisms may play a role in their clinical efficacy.     

Relation between brain 5-HIAA levels and the release of serotonin into brain synapses

Our findings in experiments using reserpine, an amine releaser, and fluoxetine, a serotonin uptake blocker, indicate that the reuptake of serotonin from brain synapses precedes its transformation to 5-hydroxyindoleacetic acid (5-HIAA). Male rats were injected with reserpine or fluoxetine alone, or with fluoxetine one hour before reserpine; control animals received diluents. Reserpine lowered brain serotonin and raised brain 5-HIAA levels. Fluoxetine alone did not change serotonin levels but lowered 5-HIAA. Fluoxetine completely antagonized the reserpine-induced increase in 5-HIAA, and significantly enhanced its depletion of serotonin. In order to determine whether the ability of fluoxetine to block the rise in 5-HIAA after reserpine resulted from its effect on serotonin reuptake or from suppression of impulse flow along serotoninergic neurons, we also examined the effects of the drugs on serotonin metabolism in distal portions of acutely transected neurons (which, presumably, were no longer able to conduct impulses). No differences were noted between the responses of intact and lesioned serotoninergic neurons, indicating that fluoxetine's blockade of the rise in brain 5-HIAA results from its effect on serotonin reuptake.     

Influence of cyclic GMP on rodent aggressive behavior

Dibutyryl cGMP (0 to 100 μg), infused intraventricularly in rats and mice, produced dose-dependent increases in brain cGMP, facilitation of shock-induced rat fighting and predatory cricket-killing, and inhibition of isolation-induced mouse fighting. The changes in rat aggression with 25 μg and in mouse aggression with 50 μg were not related to sedation or motor disturbance, since locomotor activity counts were normal as were brain levels of norepinephrine, dopamine, dihydroxyphenylacetic acid, serotonin, and 5-hydroxyindoleacetic acid. These changes seem to be related to an intracellular action of cGMP and appear to be specific for the guanine cyclic nucleotide.     

Extreme aggression in male squid induced by a β-MSP-like pheromone

Male-male aggression is widespread in the animal kingdom and subserves many functions related to the acquisition or retention of resources such as shelter, food, and mates. These functions have been studied widely in the context of sexual selection, yet the proximate mechanisms that trigger or strengthen aggression are not well known for many taxa. Various external sensory cues (visual, audio, chemical) acting alone or in combination stimulate the complex behavioral interactions of fighting behaviors. Here we report the discovery of a 10 kDa protein, termed Loligo β-microseminoprotein (Loligo β-MSP), that immediately and dramatically changes the behavior of male squid from calm swimming and schooling to extreme fighting, even in the absence of females. Females synthesize Loligo β-MSP in their reproductive exocrine glands and embed the protein in the outer tunic of egg capsules, which are deposited on the open sea floor. Males are attracted to the eggs visually, but upon touching them and contacting Loligo β-MSP, they immediately escalate into intense physical fighting with any nearby males. Loligo β-MSP is a distant member of the chordate β-microseminoprotein family found in mammalian reproductive secretions, suggesting that this gene family may have taxonomically widespread roles in sexual competition.     

Long-term fluoxetine administration does not result in major changes in bone architecture and strength in growing rats

Many studies have indicated that serotonin and its transporter play a role in bone metabolism. In this study we investigated the effect of selective serotonin re-uptake inhibitor (SSRI), fluoxetine (Prozac) on bone architecture and quality in growing female rats. We therefore administrated rats with clinically relevant doses of fluoxetine for a period of 6 months. DXA scans were performed during the treatment period in order to follow parameters as body weight, fat percentage and BMD. After 6 months of treatment, femurs were used to analyze bone architecture and bone strength, by means of microCT scans and three-point bending assays, respectively. We found a slightly diminished bone quality, reflected in a lower bone tissue strength, which was compensated by changes in bone geometry. As leptin and adiponectin could be possible factors in the serotonergic regulation of bone metabolism, we also determined the levels of these factors in plasma samples of all animals. Leptin and adiponectin levels were not different between the control group and fluoxetine-treated group, indicating that these factors were not involved in the observed changes in bone geometry and quality.     

Novelty stress and reproductive state alters responsiveness to sensory stimuli and 5-HT neuromodulation in crayfish

Sensory stimuli can produce varied responses depending on the physiological state of an animal. Stressors and reproductive stage can result in altered biochemical status that changes the responsiveness of an animal to hormones and neuromodulators, which affects whole animal behavior in relation to sensory stimuli. Crayfish serve as a model for examining the effects of neuromodulators at the neuromuscular junctions (NMJs) and for alterations in stereotypic behaviors for particular stimuli. Thus, we used crayfish to examine the effect of novelty stressors in males and the effect of being gravid in female crayfish to exogenous application of serotonin (5-HT). The responsiveness of neuromuscular junctions to 5-HT revealed that stressed as well as gravid crayfish have a reduced response to 5-HT at NMJs. The stressed crayfish were not fatigued since the basal synaptic responses are large and still showed a pronounced response to 5-HT. Using intact animals to examine a tail flip behavior, we showed that the rate of habituation in tail flipping to a strong repetitive stimulus on the telson is reduced in stressed males. Gravid females show no tail flipping behavior upon telson stimulation.