Fingerprinting of Psychoactive Drugs in Zebrafish Anxiety-Like Behaviors

A major hindrance for the development of psychiatric drugs is the prediction of how treatments can alter complex behaviors in assays which have good throughput and physiological complexity. Here we report the development of a medium-throughput screen for drugs which alter anxiety-like behavior in adult zebrafish. The observed phenotypes were clustered according to shared behavioral effects. This barcoding procedure revealed conserved functions of anxiolytic, anxiogenic and psychomotor stimulating drugs and predicted effects of poorly characterized compounds on anxiety. Moreover, anxiolytic drugs all decreased, while anxiogenic drugs increased, serotonin turnover. These results underscore the power of behavioral profiling in adult zebrafish as an approach which combines throughput and physiological complexity in the pharmacological dissection of complex behaviors.     

Relation between the nature of psychotropic effects on emotional reactivity and behavior in stress situations and the status of the brain's catecholaminergic systems

Effects of psychotropic drugs on emotional reactivity and behavior under acute stress were studied in rats with 6-hydroxydopamine (6-HDA)-destroyed catecholaminergic brain terminals. The differences in emotional and behavioral reactivity were found in the group of animals who received 6-HDA. An abrupt reduction of tyrosine hydroxylase activity in corpus striatum of "emotional" rats correlates with noticeable difficulties in the behavior of escape out of stress and essential differences in the effects of psychotropic drugs whose action is mediated via the catecholamine neurotransmitter system.     

Interaction of the tachykinin NK3 receptor agonist senktide with behavioral effects of cocaine in marmosets (Callithrix penicillata)

Brain neuropeptide transmitters of the tachykinin family are involved in the organization of many behaviors. However, little is known about their contribution to the behavioral effects of drugs of abuse. Recently, antagonism of the tachykinin NK3-receptor (NK3-R), one of the three tachykinin receptors in the brain, was shown to attenuate the acute and chronic behavioral effects of cocaine in rats and the acute effects in non-human primates. In order to expand these findings we investigated the effects of the NK3-R agonist, succinyl-[Asp6, Me-Phe8]SP(6-11) (senktide), on the acute behavioral effects of cocaine in marmoset monkeys (Callithrix penicillata) using a figure-eight maze procedure. Animals were pretreated with senktide (0, 0.1, 0.2, 0.4 mg/kg, s.c.), and received either a treatment with cocaine (10 mg/kg) or saline (i.p.). Cocaine increased locomotor activity and the duration of aerial scanning behavior, but reduced exploratory activity, bodycare activity, the frequency of aerial scanning, and terrestrial glance behavior. Senktide blocked the effects of cocaine on locomotor activity, but enhanced the cocaine effects on exploratory activity, aerial scanning frequency, and terrestrial glance behavior. Senktide alone did not significantly influence monkey behavior in this study. These data expand previous findings suggesting a complex role of the NK3-R in the acute behavioral effects of cocaine in non-human primates.     

The Use of Genetic “Knockout” Mice in Behavioral Endocrinology Research

The production of mice with specific deletion of targeted genes (knockouts) has provided a useful tool in understanding the mechanisms underlying behavior. There are many opportunities with this new tool for behavioral neuroendocrinology, specifically, and behavioral biology, generally. Although this genetic technique offers new opportunities to study the mechanisms of behavior, as with all behavioral techniques there are some potential limitations. For example, the products of many genes are essential to normal function, and inactivating the gene may prove lethal or induce gross morphological or physiological abnormalities that can complicate interpretation of discrete behavioral effects. Unexpected compensatory or redundancy mechanisms might be activated when a gene is missing and cloud interpretation of the normal contribution of the gene to behavior. Behavioral tests study the effects of themissinggene (and gene product), not the effects of the gene directly. This conceptual shortcoming can be overcome in the same way that it is overcome in other types of ablation studies, by collecting converging evidence using a variety of pharmacological, lesion, and genetic manipulations. Finally, because mammalian genome mapping is currently focused on mice (Mus musculus), standardized behavioral testing of mice should be adopted. Against those disadvantages are several important advantages to using knockout mice in behavioral research: (1) disabling a gene is often a very precise and “clean” ablation, (2) the effects of the gene product can be abolished without the side-effects of drugs, and (3) genetic manipulations may be the only way to determine the precise role of many endogenous factors on behavior. The use of new inducible knockouts, in which the timing and placement of the targeted gene disruption can be controlled, will be an extremely important tool in behavioral endocrinology research.     

Impulsivity as a determinant and consequence of drug use: a review of underlying processes

Impulsive behaviors are closely linked to drug use and abuse, both as contributors to use and as consequences of use. Trait impulsivity is an important determinant of drug use during development, and in adults momentary 'state' increases in impulsive behavior may increase the likelihood of drug use, especially in individuals attempting to abstain. Conversely, acute and chronic effects of drug use may increase impulsive behaviors, which may in turn facilitate further drug use. However, these effects depend on the behavioral measure used to assess impulsivity. This article reviews data from controlled studies investigating different measures of impulsive behaviors, including delay discounting, behavioral inhibition and a newly proposed measure of inattention. Our findings support the hypothesis that drugs of abuse alter performance across independent behavioral measures of impulsivity. The findings lay the groundwork for studying the cognitive and neurobiological substrates of impulsivity, and for future studies on the role of impulsive behavior as both facilitator and a result of drug use.     

GABA and the behavioral effects of anxiolytic drugs

Much recent research has shown that benzodiazepine binding sites in the central nervous system are associated with GABA receptors. It is therefore possible that the pharmacological and therapeutic effects of benzodiazepines and drugs with similar profiles are mediated through GABAergic mechanisms. In this paper the evidence is considered for a possible involvement of GABA in the behavioral effects of anxiolytic drugs. There are a number of reports that the behavioral actions of anxiolytics can be antagonised by GABA antagonists such as bicuculline or picrotoxin but there are many contradictory findings and these drugs are difficult to use effectively in behavioral studies. In general, GABA agonists do not exert anxiolytic-like behavioral effects after systemic injection but intracerebral administration of muscimol has been shown to produce benzodiazepine-like actions. Although a number of questions remain unanswered, current evidence does not provide strong support for a role for GABA in the behavioral effects of anxiolytic drugs.     

Potentiation of the behavioral effects of pentobarbital, chlordiazepoxide and ethanol by thyrotropin-releasing hormone

Effects of pentobarbital, chlordiazepoxide and ethanol were studied alone and in combination with thyrotropin-releasing hormone (TRH), IM, on punished behavior. Key-peck responses of pigeons were maintained by food presentation under a fixed-interval 3-min schedule in which every 30th response produced shock. Moderate doses of pentobarbital, chlordiazepoxide and ethanol increased punished responding to 150-200% of control values while the higher doses of these drugs almost completely eliminated responding. TRH (0.01-1 mg/kg) had little effect on punished responding and 3 mg/kg produced 50% decreases. Although the lower doses of TRH were without effect when given alone, doses of 0.03 mg/kg and greater markedly potentiated the rate-increasing effects of pentobarbital, chlordiazepoxide and ethanol. Increases in punished responding of 350% were obtained with combinations of TRH and these drugs. The rate-decreasing effects of the sedative-hypnotic and anxiolytic compounds were not reversed by TRH. Potentiation of the behavioral effects of sedative-hypnotic and anxiolytic drugs by TRH suggests that TRH may play an important role in modulating the behavioral effects of these compounds and that combinations of neuroactive peptides with certain psychotherapeutic agents may be of some therapeutic value.     

Invertebrate models of drug abuse

Susceptibility to drug addiction depends on genetic and environmental factors and their complex interactions. Studies with mammalian models have identified molecular targets, neurochemical systems, and brain regions that mediate some of the addictive properties of abused drugs. Yet, our understanding of how the primary effects of drugs lead to addiction remains incomplete. Recently, researchers have turned to the invertebrate model systems Drosophila melanogaster and Caenorhabditis elegans to dissect the mechanisms by which abused drugs modulate behavior. Due to their sophisticated genetics, relatively simple anatomy, and their remarkable molecular similarity to mammals, these invertebrate models should provide useful insights into the mechanisms of drug action. Here we review recent behavioral and genetic studies in flies and worms on the effects of ethanol, cocaine, and nicotine, three of the most widely abused drugs in the world.     

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.     

Integrating synaptic plasticity and striatal circuit function in addiction

Exposure to addictive drugs causes changes in synaptic function within the striatal complex, which can either mimic or interfere with the induction of synaptic plasticity. These synaptic adaptations include changes in the nucleus accumbens (NAc), a ventral striatal subregion important for drug reward and reinforcement, as well as the dorsal striatum, which may promote habitual drug use. As the behavioral effects of drugs of abuse are long-lasting, identifying persistent changes in striatal circuits induced by in vivo drug experience is of considerable importance. Within the striatum, drugs of abuse have been shown to induce modifications in dendritic morphology, ionotropic glutamate receptors (iGluR) and the induction of synaptic plasticity. Understanding the detailed molecular mechanisms underlying these changes in striatal circuit function will provide insight into how drugs of abuse usurp normal learning mechanisms to produce pathological behavior.     

Ergot drugs and central monoaminergic mechanisms: a histochemical, biochemical and behavioral analysis.

The effects of ergot drugs on central monoaminergic mechanisms, particularly dopaminergic mechanisms, have been investigated in histochemical, biochemical and behavioral studies. It is concluded that the behavioral effects and the antiparkinsonian properties of ergot drugs can to a large extent be explained by the hypothesis that ergot drugs act at various dopamine receptor sites in the brain as partial agonists, the ratio of agonist-antagonist activity varying from one dopamine receptor population to another.     

Drosophila melanogaster as a model to study drug addiction

Animal studies have been instrumental in providing knowledge about the molecular and neural mechanisms underlying drug addiction. Recently, the fruit fly Drosophila melanogaster has become a valuable system to model not only the acute stimulating and sedating effects of drugs but also their more complex rewarding properties. In this review, we describe the advantages of using the fly to study drug-related behavior, provide a brief overview of the behavioral assays used, and review the molecular mechanisms and neural circuits underlying drug-induced behavior in flies. Many of these mechanisms have been validated in mammals, suggesting that the fly is a useful model to understand the mechanisms underlying addiction.     

Castration and antisteroid treatment impair vocal learning in male zebra finches.

Both song behavior and its neural substrate are hormone sensitive: castrated adult male zebra finches need replacement of gonadal steroids in order to restore normal levels of song production, and sex steroids are necessary to establish male-typical neural song-control circuits during early development. This pattern of results suggests that hormones may be required for normal development of learned song behavior, but evidence that steroids are necessary for normal neural and behavioral development during song learning has been lacking. We addressed this question by attempting to eliminate the effects of gonadal steroids in juvenile male zebra finches between the time of initial song production and adulthood. Males were castrated at 20 days of age and received systemic implants of either an antiandrogen (flutamide), an antiestrogen (tamoxifen), or both drugs. The songs of both flutamide- and tamoxifen-treated birds were extremely disrupted relative to normal controls in terms of the stereotypy and acoustic quality of individual note production, as well as stereotypy of the temporal structure of the song phrase. We did not discern any differences in the pattern of behavioral disruption between birds that were treated with either flutamide, tamoxifen, or a combination of both drugs. Flutamide treatment resulted in a reduced size of two forebrain nuclei that are known to play some role unique to early phases of song learning [lateral magnocellular nucleus of the anterior neostriatum (IMAN) and area X (X)], but did not affect the size of two song-control nuclei that are necessary for normal song production in adult birds [caudal nucleus of the ventral hyperstriatum (HVc) and robust nucleus of the archistriatum (RA)]. In contrast, treatment with tamoxifen did not result in any changes in the size of song-control nuclei relative to normal controls, and it blocked the effects of flutamide on the neural song-control system in birds that were treated with both drugs. Castration and antisteroid treatment exerted no deleterious effects on the quality of song behavior in adult birds, indicating that gonadal hormones are necessary for the development of normal song behavior during a sensitive period.     

Waterborne Risperidone Decreases Stress Response in Zebrafish

The presence of drugs and their metabolites in surface waters and municipal effluents has been reported in several studies, but its impacts on aquatic organisms are not yet well understood. This study investigated the effects of acute exposure to the antipsychotic risperidone on the stress and behavioral responses in zebrafish. It became clear that intermediate concentration of risperidone inhibited the hypothalamic-pituitary-interrenal axis and displayed anxiolytic-like effects in zebrafish. The data presented here suggest that the presence of this antipsychotic in aquatic environments can alter neuroendocrine and behavior profiles in zebrafish.     

Why Evolutionary Issues are Reviving Interest in Proximate Behavioral Mechanisms

Information about proximate behavioral mechanisms is requiredto answer a variety of important questions about the evolutionof behavioral and other traits. For instance, theoretical studiesof maternal effects indicate that the behavior of parents mayhave profound effects on the evolution of phenotypic traitsin their offspring, and that maternal effects may provide nongeneticpathways for the inheritance of both behavioral and nonbehavioraltraits. Similarly, the recent emphasis on genetic correlationsamong traits cautions that correlations among behavioral traitsmay have surprising effects on behavioral evolution, and providesfurther impetus for studies of the proximate bases of behavioraltrait syndromes. Finally, the literature on information acquisitionsuggests that the behavioral processes used in assessment mayaffect the evolution of behavioral and other traits. For instance,if information is costly to obtain, individuals may prefer familiarto unfamiliar situations. In a social context, a preferencefor familiar individuals could have a variety of effects onthe evolution of social and nonsocial behavior patterns. Theseand other examples suggest that interest in behavioral processeswill continue to grow during the next decade, and that proximateand ultimate approaches to behavioral problems will be moreclosely integrated than was true in the past.     

Evidence for the involvement of ERbeta and RGS9-2 in 17-beta estradiol enhancement of amphetamine-induced place preference behavior

Estrogen enhances dopamine-mediated behaviors, which make women and female rats more sensitive to the effects of the psychostimulant drugs, cocaine and amphetamine. How cocaine and amphetamine elicit more robust behavioral responses in females remains unclear, but studies have shown that the Regulator of G-protein Signaling 9-2 (RGS9-2) protein is an important modulator of the behavioral responses to these drugs. Previously, we reported that 17-beta estradiol reduced RGS9-2 mRNA expression in the shell of the nucleus accumbens, but not the core. The present studies were designed to further evaluate the involvement of RGS9-2 in estradiol enhancement of amphetamine-induced place preference behavior and to examine which estrogen receptor subtype mediates the effect of estradiol. Female Sprague-Dawley rats were ovariectomized and treated for 14 days with an inert vehicle or 17-beta estradiol (by Silastic implant or injection [80 microg/kg]). 17-beta-Estradiol-treated female rats had enhanced amphetamine-induced conditioned place preference behavior compared to vehicle-treated, ovariectomized female rats. In situ hybridization histochemistry and Western blotting identified an inverse relationship between RGS9-2 protein expression in the nucleus accumbens shell and the hormonal enhancement of amphetamine-induced place preference behavior. A similar relationship was not found between place preference behavior and RGS9-2 expression in the accumbens core. Moreover, treatment of ovariectomized female rats with the selective estrogen receptor-beta agonist, diarylpropionitrile (1 mg/kg), for 2 weeks also facilitated amphetamine-induced place preference behavior and selectively reduced nucleus accumbens shell RGS9-2 protein expression. These data provide insight into a potential mechanism by which estrogen and/or sex modulate mesoaccumbal dopamine receptor signaling and possibly, addictive behaviors.     

“Sex, drugs and the brain”: The interaction between drugs of abuse and sexual behavior in the female rat

Preclinical and clinical research investigating female sexual motivation has lagged behind research on male sexual function. The present review summarizes recent advances in our understanding of the specific roles of various brain areas, as well as our understanding of the role of dopaminergic neurotransmission in sexual motivation of the female rat. A number of behavioral paradigms that can be used to thoroughly evaluate sexual behavior in the female rat are first discussed. Although traditional assessment of the reflexive, lordosis posture has been useful in understanding the neuroanatomical and neurochemical systems that contribute to copulatory behavior, the additional behavioral paradigms described in this review have helped us expand our understanding of appetitive and consumatory behavioral patterns that better assess sexual motivation - the equivalent of “desire” in humans. A summary of numerous lesion studies indicates that different areas of the brain, including forebrain and midbrain structures, work together to produce the complex repertoire of female sexual behavior. In addition, by investigating the effects of commonly addictive drugs, we are beginning to elucidate the role of dopaminergic neurotransmission in female sexual motivation. Consequently, research in this area may contribute to meaningful advances in the treatment of human female sexual dysfunction.     

Behavioral screening for neuroactive drugs in zebrafish

The larval zebrafish has emerged asa vertebrate model system amenable to small molecule screens for probing diverse biological pathways. Two large-scale small molecule screens examined the effects of thousands of drugs on larval zebrafish sleep/wake and photomotor response behaviors. Both screens identified hundreds of molecules that altered zebrafish behavior in distinct ways. The behavioral profiles induced by these small molecules enabled the clustering of compounds according to shared phenotypes. This approach identified regulators of sleep/wake behavior and revealed the biological targets for poorly characterized compounds. Behavioral screening for neuroactive small molecules in zebrafish is an attractive complement to in vitro screening efforts, because the complex interactions in the vertebrate brain can only be revealed in vivo.     

Differential interaction between stressors and chronic amphetamine or phencyclidine upon operant behavior in the rat

Three types of stress were examined for their effects upon fixed-ratio 15 (FR-15) operant behavior in Sprague-Dawley rats before and after chronic dextroamphetamine to determine if cumulated drug could interact with stress to produce a state of behavioral toxicity. Six daily s.c. injections of saline were given to rats following 30-min behavioral sessions. Thirty minutes of exposure to a cold environment (7°C) or to electric footshock (2mA for 0.5 sec, 1 shock/min) had no significant effect upon FR-15 behavior beginning 15 min afterward. Five daily s.c. injections of 2.5 mg dextroamphetamine/kg after behavioral sessions suppressed behavior during the following days an average of 12%. Rats were again stressed twenty-two hours after the sixth injection. Footshock further suppressed behavior to 26% of the unstressed, chronic amphetamine-treated control group. Cold exposure failed to suppress behavior significantly below control levels. A similar experiment employing cold water exposure (15°C for 2 min) as the stressor showed that, although this stressor suppressed FR-15 behavior to 53% of the previous day's rates and reduced body temperature 2.1°C, chronic amphetamine failed to interact with the cold water stress to suppress behavior further. In a third experiment, 4.5 mg phencyclidine/kg, s.c., given after 30-min FR-15 sessions for six days, failed to interact with the footshock stress to cause behavioral suppression different from control treatment. While stress can interact with chronic treatment with lipophilic drugs, the quality of the stress, and the physical and pharmacologic properties of the drug are important determinants of the outcome upon conditioned behavior.     

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.