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.     

The benzodiazepine receptor inverse agonists beta-CCM and RO 15-3505 both reverse the anxiolytic effects of ethanol in mice

The antagonistic effects of the benzodiazepine receptor inverse agonist beta-CCM (1 mg/kg) and of the partial inverse agonist RO 15-3505 (3 mg/kg) on the anxiolytic properties of ethanol (1 g/kg) in mice confronted with a light/dark choice procedure and with the staircase test were investigated. Both drugs reversed the effects of ethanol on some of the behavioral parameters, but beta-CCM alone elicited anxiogenic intrinsic effects. RO 15-3505 induced seizures in mice treated with a subconvulsant dose of pentylenetetrazole, the most efficient doses being 3 and 6 mg/kg. These data indicate that beta-CCM and RO 15-3505 can reverse some of the anxiolytic effects of ethanol, acting probably to oppose GABA function via the benzodiazepine receptor.     

The behavioral response of zebrafish to hypergravity conditions

Previous reports of the behavior of aquatic organisms in the microgravity environment of space (~10(-6) g) or during the brief weightless period of parabolic flight indicate that most species display a dramatic "looping" or "circling" response (De Jong et al. 1996, Anken, Ibsch and Rahmann 1998). However, the behavior of aquatic species under hypergravity conditions is less clear. Our objectives in the present study were to examine the behavioral response of adult zebrafish (Danio rerio) to hypergravity conditions (2-g), quantify changes in adult swimbladder volume, and to determine if the larvae of zebrafish are capable of accessing the air-water interface for initial swimbladder inflation under hypergravity conditions.     

A change in the alarm level entails a change in behavioural strategy of mice in stress and a change in analgesia induced by it

The effects of anxiogenic (pentylentetrazole) and anxiolytic (diazepam) agents on and cold swim stress-induced analgesia were investigated in SHR and NMRI male mice. It was shown that behavioral response to acute stress was associated with a change in the pain tolerance threshold. Diazepam increased immobility time and attenuated stress-induced analgesia (SIA). NMRI mice were more responsive to anxiolytic than the SHR mice, but the lattes manifested more dramatic changes when anxiety was pharmacologically enhanced (immobility time was significantly reduced and the SIA exaggerated). Our findings suggest that the main parameters change in reciprocal manner following a pharmacologically altered anxiety, and reveal that differences between two strains of mice are determined by differences in their sensitivity to stress.     

Seizures Induced by Pentylenetetrazole in the Adult Zebrafish: A Detailed Behavioral Characterization

Pentylenetetrazole (PTZ) is a common convulsant agent used in animal models to investigate the mechanisms of seizures. Although adult zebrafish have been recently used to study epileptic seizures, a thorough characterization of the PTZ-induced seizures in this animal model is missing. The goal of this study was to perform a detailed temporal behavior profile characterization of PTZ-induced seizure in adult zebrafish. The behavioral profile during 20 min of PTZ immersion (5, 7.5, 10, and 15 mM) was characterized by stages defined as scores: (0) short swim, (1) increased swimming activity and high frequency of opercular movement, (2) erratic movements, (3) circular movements, (4) clonic seizure-like behavior, (5) fall to the bottom of the tank and tonic seizure-like behavior, (6) death. Animals exposed to distinct PTZ concentrations presented different seizure profiles, intensities and latencies to reach all scores. Only animals immersed into 15 mM PTZ showed an increased time to return to the normal behavior (score 0), after exposure. Total mortality rate at 10 and 15 mM were 33% and 50%, respectively. Considering all behavioral parameters, 5, 7.5, 10, and 15 mM PTZ, induced seizures with low, intermediate, and high severity, respectively. Pretreatment with diazepam (DZP) significantly attenuated seizure severity. Finally, the brain PTZ levels in adult zebrafish immersed into the chemoconvulsant solution at 5 and 10 mM were comparable to those described for the rodent model, with a peak after a 20-min of exposure. The PTZ brain levels observed after 2.5-min PTZ exposure and after 60-min removal from exposure were similar. Altogether, our results showed a detailed temporal behavioral characterization of a PTZ epileptic seizure model in adult zebrafish. These behavioral analyses and the simple method for PTZ quantification could be considered as important tools for future investigations and translational research.     

Chronic stress and forming of psychoemotional state during prepubertal period in rats

Immediate and long-lasting effects of chronic stress during prepubertal period (21-32 postnatal days) on anxiety- and depression-related behavior were studied in Wistar and ISIAH (inherited stress-induced arterial hypertension) rats. Significant interstrain differences were found. Both juvenile and adult ISIAH rats were less anxious in the elevated plus-maze and less depressed in the forced swimming test. Immediate effects of the prepubertal stress were similar in both rat strains and depended on the type of stimulation. Long-lasting effects were genotype-dependent. Chronic prepubertal handling exerted an anxiolytic effect in young ISIAH and Wistar rats and adult Wistar rats. Immediate anxiogenic effect of prepubertal unpredictable stress was preserved only in adult ISIAH rats. Depression-related behavior was intensified by the unpredictable stress in young animals, whereas the long-lasting effect was observed only in adult hypertensive rats.     

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.     

Arsenic alters behavioral parameters and brain ectonucleotidases activities in zebrafish (Danio rerio)

Arsenic (As) exposure has been associated with serious chronic health risk to humans including cancer and neurological disturbances. However, there are limited studies about the mechanisms behind its toxicity. In this study, adult zebrafish were exposed to several concentrations of As (0.05, 5, and 15 mg As/L; Na(2)HAsO(4) as As(V)) during 96 h to evaluate the zebrafish locomotor activity, anxiety, and brain extracellular nucleotide hydrolysis. We showed that 5 mg/L As is able to promote significant decrease in the locomotor activity as evaluated by the number of line crossings. In addition, animals treated with 5mg/L As presented an increase in time spent in the lower zone of the tank test, suggesting an anxiogenic effect. Considering that behavioral parameters, such as anxiety and locomotion, might be modulated by the purinergic system, we also evaluated the ectonucleotidase activities in zebrafish brain after a 96-h As exposure. A significant decrease in ATP, ADP, and AMP hydrolysis was observed at 0.05, 5, and 15 mg/L when compared to control group. These findings demonstrated that As might affect behavioral parameters and the ectonucleotidase activities in zebrafish, suggesting this enzyme pathway is a target for neurotoxic effects induced by As.     

Neonatal cerebral cortical lesion abolishes the anxiolytic action of diazepam in adult rats: effects of location and extent of cortical lesion.

In our previous study, diazepam (DZP), a benzodiazepine receptor agonist, failed to suppress foot-shock-elicited ultrasonic vocalizations (USVs) in adult rats that had been neonatally lesioned in the neocortex. Because neonatal lesion of the neocortex did not influence the production of USVs, the presence of an anxiolytic mechanism of DZP is suggested apart from any anxiogenic mechanism in the brain. However, the previous study did not indicate any specific cortical regional lesions that impaired the normal development of the anxiolytic mechanism in the brain. The present study was undertaken in order to examine whether neonatal lesion of the neocortex, smaller and more localized than that in the previous study, abolishes the anxiolytic effect of DZP on foot-shock-elicited and air-puff-elicited USVs. A neonatal lesion about 2 mm diameter was made in the unilateral frontal cortex frontal to the hindlimb area or in the occipital cortex caudal to the hindlimb area. The attenuating effect of DZP on the USVs elicited by both aversive stimuli was found to be abolished only in the frontal cortex-lesioned rats. This finding indicates that the frontal cortex is likely to be specifically involved in the normal development of the benzodiazepine-anxiolytic mechanism in the brain.     

The Difference between Anxiolytic and Anxiogenic Effects Induced by Acute and Chronic Alcohol Exposure and Changes in Associative Learning and Memory Based on Color Preference and the Cause of Parkinson-Like Behaviors in Zebrafish

We describe an interdisciplinary comparison of the effects of acute and chronic alcohol exposure in terms of their disturbance of light, dark and color preferences and the occurrence of Parkinson-like behavior in zebrafish through computer visual tracking, data mining, and behavioral and physiological analyses. We found that zebrafish in anxiolytic and anxious states, which are induced by acute and chronic repeated alcohol exposure, respectively, display distinct emotional reactions in light/dark preference tests as well as distinct learning and memory abilities in color-enhanced conditional place preference (CPP) tests. Additionally, compared with the chronic alcohol (1.0%) treatment, acute alcohol exposure had a significant, dose-dependent effect on anxiety, learning and memory (color preference) as well as locomotive activities. Acute exposure doses (0.5%, 1.0%, and 1.5%) generated an “inverted V” dose-dependent pattern in all of the behavioral parameters, with 1.0% having the greatest effect, while the chronic treatment had a moderate effect. Furthermore, by measuring locomotive activity, learning and memory performance, the number of dopaminergic neurons, tyrosine hydroxylase expression, and the change in the photoreceptors in the retina, we found that acute and chronic alcohol exposure induced varying degrees of Parkinson-like symptoms in zebrafish. Taken together, these results illuminated the behavioral and physiological mechanisms underlying the changes associated with learning and memory and the cause of potential Parkinson-like behaviors in zebrafish due to acute and chronic alcohol exposure.     

Selective effect on punished versus unpunished responding in a conflict test as the criterion for anxiogenic activity

The punished drinking test has been used successfully for identifying and studying anxiolytic agents. By reducing the level of punishment (i.e., decreasing the intensity of shock), it has also been used as a method for measuring anxiogenic activity. Because anxiogenic behavior is a novel and important concept that is not yet fully established, we have reinvestigated the effects of two putative inverse benzodiazepine agonists and pentylenetetrazol in this conflict test. In a series of experiments, using both our version of the procedure and a replication of a previously published method, we were unable to demonstrate a selective reduction in punished responding over unpunished responding caused by CGS 8216 (3 to 40 mg/kg), FG 7142 (2 to 6 mg/kg), and pentylenetetrazol (10 to 20 mg/kg) as reported previously. A careful comparison of the details of our method and the published procedure failed to reveal the source of this discrepancy. If anxiogenic behavior is to be defined as a selective effect of a drug on punished response, the value of this test will depend on identification of its critical variables.     

Measuring behavioral and endocrine responses to novelty stress in adult zebrafish

Several behavioral assays are currently used for high-throughput neurophenotyping and screening of genetic mutations and psychotropic drugs in zebrafish (Danio rerio). In this protocol, we describe a battery of two assays to characterize anxiety-related behavioral and endocrine phenotypes in adult zebrafish. Here, we detail how to use the 'novel tank' test to assess behavioral indices of anxiety (including reduced exploration, increased freezing behavior and erratic movement), which are quantifiable using manual registration and computer-aided video-tracking analyses. In addition, we describe how to analyze whole-body zebrafish cortisol concentrations that correspond to their behavior in the novel tank test. This protocol is an easy, inexpensive and effective alternative to other methods of measuring stress responses in zebrafish, thus enabling the rapid acquisition and analysis of large amounts of data. As will be shown here, fish anxiety-like behavior can be either attenuated or exaggerated depending on stress or drug exposure, with cortisol levels generally expected to parallel anxiety behaviors. This protocol can be completed over the course of 2 d, with a variable testing duration depending on the number of fish used.     

Sex and exercise interact to alter the expression of anabolic androgenic steroid-induced anxiety-like behaviors in the mouse

Anabolic androgenic steroids (AAS) are taken by both sexes to enhance athletic performance and body image, nearly always in conjunction with an exercise regime. Although taken to improve physical attributes, chronic AAS use can promote negative behavior, including anxiety. Few studies have directly compared the impact of AAS use in males versus females or assessed the interaction of exercise and AAS. We show that AAS increase anxiety-like behaviors in female but not male mice and that voluntary exercise accentuates these sex-specific differences. We also show that levels of the anxiogenic peptide corticotrophin releasing factor (CRF) are significantly greater in males, but that AAS selectively increase CRF levels in females, thus abrogating this sex-specific difference. Exercise did not ameliorate AAS-induced anxiety or alter CRF levels in females. Exercise was anxiolytic in males, but this behavioral outcome did not correlate with CRF levels. Brain-derived neurotrophic factor (BDNF) has also been implicated in the expression of anxiety. As with CRF, levels of hippocampal BDNF mRNA were significantly greater in males than females. AAS and exercise were without effect on BDNF mRNA in females. In males, anxiolytic effects of exercise correlated with increased BDNF mRNA, however AAS-induced changes in BDNF mRNA and anxiety did not. In sum, we find that AAS elicit sex-specific differences in anxiety and that voluntary exercise accentuates these differences. In addition, our data suggest that these behavioral outcomes may reflect convergent actions of AAS and exercise on a sexually differentiated CRF signaling system within the extended amygdala.     

Anxiogenic effects of developmental bisphenol a exposure are associated with gene expression changes in the juvenile rat amygdala and mitigated by soy

Early life exposure to Bisphenol A (BPA), a component of polycarbonate plastics and epoxy resins, alters sociosexual behavior in numerous species including humans. The present study focused on the ontogeny of these behavioral effects beginning in adolescence and assessed the underlying molecular changes in the amygdala. We also explored the mitigating potential of a soy-rich diet on these endpoints. Wistar rats were exposed to BPA via drinking water (1 mg/L) from gestation through puberty, and reared on a soy-based or soy-free diet. A group exposed to ethinyl estradiol (50 μg/L) and a soy-free diet was used as a positive estrogenic control. Animals were tested as juveniles or adults for anxiety-like and exploratory behavior. Assessment of serum BPA and genistein (GEN), a soy phytoestrogen, confirmed that internal dose was within a human-relevant range. BPA induced anxiogenic behavior in juveniles and loss of sexual dimorphisms in adult exploratory behavior, but only in the animals reared on the soy-free diet. Expression analysis revealed a suite of genes, including a subset known to mediate sociosexual behavior, associated with BPA-induced juvenile anxiety. Notably, expression of estrogen receptor beta (Esr2) and two melanocortin receptors (Mc3r, Mc4r) were downregulated. Collectively, these results show that behavioral impacts of BPA can manifest during adolescence, but wane in adulthood, and may be mitigated by diet. These data also reveal that, because ERβ and melanocortin receptors are crucial to their function, oxytocin/vasopressin signaling pathways, which have previously been linked to human affective disorders, may underlie these behavioral outcomes.     

Acute and chronic alcohol dose: population differences in behavior and neurochemistry of zebrafish

The zebrafish has been in the forefront of developmental genetics for decades and has also been gaining attention in neurobehavioral genetics. It has been proposed to model alcohol-induced changes in human brain function and behavior. Here, adult zebrafish populations, AB and SF (short-fin wild type), were exposed to chronic treatment (several days in 0.00% or 0.50% alcohol v/v) and a subsequent acute treatment (1 h in 0.00%, 0.25%, 0.50% or 1.00% alcohol). Behavioral responses of zebrafish to computer-animated images, including a zebrafish shoal and a predator, were quantified using videotracking. Neurochemical changes in the dopaminergic and serotoninergic systems in the brain of the fish were measured using high-precision liquid chromatography with electrochemical detection. The results showed genetic differences in numerous aspects of alcohol-induced changes, including, for the first time, the behavioral effects of withdrawal from alcohol and neurochemical responses to alcohol. For example, withdrawal from alcohol abolished shoaling and increased dopamine and 3,4-dihydroxyphenylacetic acid in AB but not in SF fish. The findings show that, first, acute and chronic alcohol induced changes are quantifiable with automated behavioral paradigms; second, robust neurochemical changes are also detectable; and third, genetic factors influence both alcohol-induced behavioral and neurotransmitter level changes. Although the causal relationship underlying the alcohol-induced changes in behavior and neurochemistry is speculative at this point, the results suggest that zebrafish will be a useful tool for the analysis of the biological mechanisms of alcohol-induced functional changes in the adult brain.     

Convolutional neural networks for reconstruction of undersampled optical projection tomography data applied to in vivo imaging of zebrafish

Optical projection tomography (OPT) is a 3D mesoscopic imaging modality that can utilize absorption or fluorescence contrast. 3D images can be rapidly reconstructed from tomographic data sets sampled with sufficient numbers of projection angles using the Radon transform, as is typically implemented with optically cleared samples of the mm‐to‐cm scale. For in vivo imaging, considerations of phototoxicity and the need to maintain animals under anesthesia typically preclude the acquisition of OPT data at a sufficient number of angles to avoid artifacts in the reconstructed images. For sparse samples, this can be addressed with iterative algorithms to reconstruct 3D images from undersampled OPT data, but the data processing times present a significant challenge for studies imaging multiple animals. We show here that convolutional neural networks (CNN) can be used in place of iterative algorithms to remove artifacts—reducing processing time for an undersampled in vivo zebrafish dataset from 77 to 15 minutes. We also show that using CNN produces reconstructions of equivalent quality to compressed sensing with 40% fewer projections. We further show that diverse training data classes, for example, ex vivo mouse tissue data, can be used for CNN‐based reconstructions of OPT data of other species including live zebrafish.      

A Robotics-Based Behavioral Paradigm to Measure Anxiety-Related Responses in Zebrafish

Zebrafish are gaining momentum as a laboratory animal species for the study of anxiety-related disorders in translational research, whereby they serve a fundamental complement to laboratory rodents. Several anxiety-related behavioral paradigms, which rest upon the presentation of live predatorial stimuli, may yield inconsistent results due to fatigue, habituation, or idiosyncratic responses exhibited by the stimulus itself. To overcome these limitations, we designed and manufactured a fully controllable robot inspired by a natural aquatic predator (Indian leaf fish, Nandus nandus) of zebrafish. We report that this robot elicits aversive antipredatorial reactions in a preference test and that data obtained therein correlate with data observed in traditional anxiety- and fear-related tests (light/dark preference and shelter-seeking). Finally, ethanol administration (0.25; 0.50; 1.00%) exerts anxiolytic effects, thus supporting the view that robotic stimuli can be used in the analysis of anxiety-related behaviors in zebrafish.     

Anxiolytic-like effect of neuropeptide S in the rat defensive burying

Neuropeptide S (NPS) has been recently identified as the endogenous ligand of a previously orphan G-protein-coupled receptor now named NPSR. Both NPS and its receptor are expressed in the brain, where they modulate different functions. In particular, it has been demonstrated that intracerebroventricular (i.c.v.) injection of NPS in rodents increases wakefulness and promotes anxiolytic-like effects. In the present study we used the defensive burying (DB) test in rats to further investigate the action of human NPS (0.1–10 nmol, i.c.v.) on anxiety-related behaviors. Diazepam (1.5 mg/kg, i.p.) and caffeine (20 mg/kg, i.p.) were used in parallel experiments as standard anxiolytic and anxiogenic drugs, respectively. None of the tested drugs produced statistical differences in the latency to contact the probe, burying behavior latency, number of shocks received or immobility/freezing duration. Caffeine increased cumulative burying behavior and the buried bedding height in a statistically significant manner thus promoting anxiogenic like effects. Opposite results were obtained with diazepam that significantly reduced these behavioral parameters. The anxiolytic-like action of diazepam was mimicked by NPS that reduced cumulative burying behavior in a dose dependent manner. Collectively, robust anxiolytic-like effects were recorded in response to NPS in the DB test. These results are of particular interest since the outcome of this assay is marginally influenced by drug effects on locomotor activity. In conclusion, we provide further evidence that NPS evokes genuine anxiolytic-like effects in the rat; therefore NPSR selective agonists are worthy of development as innovative drugs for the treatment of anxiety disorders.     

Increased neuronal activity in locus coeruleus from adult rats undernourished at perinatal age: its reversal by desipramine.

The spontaneous activity of locus coeruleus (LC) noradrenergic neurons was assessed by single unit recording in adult recovered rats undernourished at perinatal age as compared with wellnourished animals. Locus coeruleus activity, measured by the firing rate of noradrenergic neurons and the number of spontaneously active cells/track was significantly higher in deprived rats than in controls. In addition, dose-response curves for the inhibitory LC activity of clonidine showed a shift to the right in deprived animals indicating a subsensitivity of alpha2-adrenergic autoreceptors. This fact suggests an alteration in the negative feedback mechanism mediated by somatodentritic alpha2 autoreceptors that modulate the activity of LC neurons, and may account for the behavioral alterations attributed to early undernutrition. Repeated desipramine (DMI) administration to deprived rats reduced LC activity to values comparable to controls, which were not affected after a similar treatment. These data extend to previous reports on long-lasting or permanent plastic changes in the CNS induced by early undernutrition, which may be reverted by pharmacological manipulations. In addition, these results support the hypothesis that alterations induced by early undernutrition are in the same direction as and resemble those described for patients with panic disorders. Furthermore, together with behavioral alterations and selective anxiolytic effect of DMI and other drugs with antipanic effects described in early malnourished rats, the present data support the proposal that perinatally deprived rats may be a useful model for screening drugs with potential antipanic activity.     

Dopamine receptor antagonism disrupts social preference in zebrafish: a strain comparison study

Zebrafish form shoals in nature and in the laboratory. The sight of conspecifics has been found reinforcing in zebrafish learning tasks. However, the mechanisms of shoaling, and those of its reinforcing properties, are not known. The dopaminergic system has been implicated in reward among other functions and it is also engaged by drugs of abuse as shown in a variety of vertebrates including zebrafish. The ontogenetic changes in dopamine levels and, to a lesser degree, in serotonin levels, have been found to accompany the maturation of shoaling in zebrafish. Thus, we hypothesized that the dopaminergic system may contribute to shoaling in zebrafish. To test this we employed a D1-receptor antagonist and quantified behavioral responses of our subjects using a social preference (shoaling) paradigm. We found significant reduction of social preference induced by the D1-R antagonist, SCH23390, in the AB strain of zebrafish, an alteration that was not accompanied by changes in motor function or vision. We also detected D1-R antagonist-induced changes in the level of dopamine, DOPAC, serotonin and 5HIAA, respectively, in the brain of AB zebrafish as quantified by HPLC with electrochemical detection. We found the antagonist-induced behavioral changes to be absent and the levels of these neurochemicals to be lower in another zebrafish population, SF, demonstrating naturally occurring genetic variability in these traits. We conclude that this variability may be utilized to unravel the mechanisms of social behavior in zebrafish, a line of research that may be extended to other vertebrates including our own species.