Siddharth Gaikwad

Analyzing habituation responses to novelty in zebrafish (Danio rerio)

Analysis of habituation is widely used to characterize animal cognitive phenotypes and their modulation. Although zebrafish (Danio rerio) are increasingly utilized in neurobehavioral research, their habituation responses have not been extensively investigated. Utilizing the novel tank test, we examine intra- and inter-session habituation and demonstrate robust habituation responses in adult zebrafish. Analyzing the intra-session habituation to novelty further, we also show that selected anxiogenic drugs (caffeine, pentylenetetrazole), as well as stress-inducing alarm pheromone, attenuated zebrafish habituation. Some acute anxiolytic agents, such as morphine and ethanol, while predictably reducing zebrafish anxiety, had no effects on habituation. Chronic ethanol and fluoxetine treatments improved intra-session habituation in zebrafish. In general, our study parallels literature on rodent habituation responses to novelty, and reconfirms zebrafish as a promising model for cognitive neurobehavioral research.

Three-Dimensional Neurophenotyping of Adult Zebrafish Behavior

The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior.

Characterization of behavioral and endocrine effects of LSD on zebrafish

Lysergic acid diethylamide (LSD) is a potent hallucinogenic drug that strongly affects animal and human behavior. Although adult zebrafish (Danio rerio) are emerging as a promising neurobehavioral model, the effects of LSD on zebrafish have not been investigated previously. Several behavioral paradigms (the novel tank, observation cylinder, light-dark box, open field, T-maze, social preference and shoaling tests), as well as modern video-tracking tools and whole-body cortisol assay were used to characterize the effects of acute LSD in zebrafish. While lower doses (5-100 microg/L) did not affect zebrafish behavior, 250 microg/L LSD increased top dwelling and reduced freezing in the novel tank and observation cylinder tests, also affecting spatiotemporal patterns of activity (as assessed by 3D reconstruction of zebrafish traces and ethograms). LSD evoked mild thigmotaxis in the open field test, increased light behavior in the light-dark test, reduced the number of arm entries and freezing in the T-maze and social preference test, without affecting social preference. In contrast, LSD affected zebrafish shoaling (increasing the inter-fish distance in a group), and elevated whole-body cortisol levels. Overall, our findings show sensitivity of zebrafish to LSD action, and support the use of zebrafish models to study hallucinogenic drugs of abuse.

Modeling withdrawal syndrome in zebrafish

The zebrafish (Danio rerio) is rapidly becoming a popular model species in behavioral neuroscience research. Zebrafish behavior is robustly affected by environmental and pharmacological manipulations, and can be examined using exploration-based paradigms, paralleled by analysis of endocrine (cortisol) stress responses. Discontinuation of various psychotropic drugs evokes withdrawal in both humans and rodents, characterized by increased anxiety. Sensitivity of zebrafish to drugs of abuse has been recently reported in the literature. Here we examine the effects of ethanol, diazepam, morphine and caffeine withdrawal on zebrafish behavior. Overall, discontinuation of ethanol, diazepam and morphine produced anxiogenic-like behavioral or endocrine responses, demonstrating the utility of zebrafish in translational research of withdrawal syndrome.

Pharmacological modulation of anxiety-like phenotypes in adult zebrafish behavioral models.

Zebrafish (Danio rerio) are becoming increasingly popular in neurobehavioral research. Here, we summarize recent data on behavioral responses of adult zebrafish to a wide spectrum of putative anxiolytic and anxiogenic agents. Using the novel tank test as a sensitive and efficient behavioral assay, zebrafish anxiety-like behavior can be bi-directionally modulated by drugs affecting the gamma-aminobutyric acid, monoaminergic, cholinergic, glutamatergic and opioidergic systems. Complementing human and rodent data, zebrafish drug-evoked phenotypes obtained in this test support this species as a useful model for neurobehavioral and psychopharmacological research.

Behavioral and physiological effects of acute ketamine exposure in adult zebrafish

Ketamine is a non-competitive glutamatergic antagonist used to induce sedation and analgesia. In sub-anesthetic doses, it induces hyperlocomotion, impairs memory and evokes stereotypic circling in rodents. Zebrafish (Danio rerio) emerged as a promising new animal model to screen the effects of psychotropic compounds. Here, we investigated the effects of sub-anesthetic doses of ketamine on anxiety, locomotion, habituation and social behavior of adult zebrafish. Acute 20-min exposure to 20 and 40 mg/L (but not 2 mg/L) of ketamine reduced anxiety, impaired intra-session habituation, evoked circular swimming and disrupted zebrafish shoaling. Additionally, ketamine reduced whole-body cortisol levels and elevated brain c-fos expression in zebrafish. Our findings demonstrate the sensitivity of zebrafish to behavioral and physiological effects of sub-anesthetic doses of ketamine, further supporting the utility of this species as a model for neuropharmacological research, including testing ketamine and related drugs.

Automated high-throughput neurophenotyping of zebrafish social behavior.

Zebrafish (Danio rerio) are rapidly becoming an important model organism in neuroscience research, representing an excellent species to study complex social phenotypes. Zebrafish actively form shoals, which can be used to quantify their shoaling behaviors, highly sensitive to various experimental manipulations. Recent advances in video-tracking techniques have enabled simultaneous tracking of multiple subjects, previously assessed by manual scoring of animal behavior. Here we examined the effect of group-size in the shoaling paradigm (ranging from 2 to 8 fish), and evaluated the ability of novel video-tracking tools to accurately track an entire shoal, compared to traditional manual analysis of shoaling phenotypes. To further validate our approach, the effects of the psychotropic drugs lysergic acid diethylamide (LSD) and 3,4-methlenedioxymethamphetamine (MDMA), as well as exposure to alarm pheromone, previously shown to affect zebrafish shoaling, were examined. Overall, a significant difference in group size was shown in the 2-fish vs. the 3-, 4-, 5-, 6-, 7- and 8-fish groups. Moreover, both LSD and MDMA treatments reduced shoaling (assessed by increased inter-fish distance) as well as proximity (time spent together) among fish. In contrast, exposure to alarm pheromone yielded an increase in shoaling and in proximity in a time-dependent manner. Importantly, a highly significant correlation for manual vs. automated analyses was revealed across all experiments. Collectively, this study further supports the utility of zebrafish to study social behavior, also demonstrating the capacity of video-tracking technology to assess zebrafish shoaling in a high-throughput and reliable manner.

Modeling seizure-related behavioral and endocrine phenotypes in adult zebrafish

Larval zebrafish (Danio rerio) have recently been suggested as a high-throughput experimental model of epilepsy-related pathogenetic states. Here we use adult zebrafish to study behavioral symptoms associated with drug-evoked seizures. Experimental epilepsy-like states were evoked in zebrafish by exposure for 20min to three chemoconvulsant drugs: caffeine (250mg/L; 1.3mM), pentylenetetrazole (1.5g/L; 11.0mM) and picrotoxin (100mg/L; 0.17mM). Fish behavior was analyzed using manual and video-tracking methods (Noldus Ethovision XT7). Compared to their respective controls, all three drug-treated groups showed robust seizure-like responses (hyperactivity bouts, spasms, circular and corkscrew swimming) accompanied by elevated whole-body cortisol levels (assessed by ELISA). In contrast, control fish did not display seizure-like behaviors and had significantly lower cortisol levels. Paralleling behavioral and endocrine phenotypes observed in clinical and rodent studies, our data implicates adult zebrafish as an emerging experimental model for epilepsy research.

Effects of hallucinogenic agents mescaline and phencyclidine on zebrafish behavior and physiology

Mescaline and phencyclidine (PCP) are potent hallucinogenic agents affecting human and animal behavior. As their psychotropic effects remain poorly understood, further research is necessary to characterize phenotypes they evoke in various animal models. Zebrafish (Danio rerio) are rapidly emerging as a new model organism for neuroscience research. Here, we examine the effects of mescaline (5-20mg/l) and PCP (0.5-3mg/l) in several zebrafish paradigms, including the novel tank, open field and shoaling tests. Mescaline and PCP dose-dependently increased top activity in the novel tank test, also reducing immobility and disrupting the patterning of zebrafish swimming, as assessed by ethograms. PCP, but not mescaline, evoked circling behavior in the open field test. At the highest doses tested, mescaline markedly increased, while PCP did not affect, zebrafish shoaling behavior. Finally, 20mg/l mescaline did not alter, and 3mg/l PCP elevated, whole-body cortisol levels. Overall, our studies indicate high sensitivity of zebrafish models to hallucinogenic compounds with complex behavioral and physiological effects.

The effects of chronic social defeat stress on mouse self-grooming behavior and its patterning

Stress induced by social defeat is a strong modifier of animal anxiety and depression-like phenotypes. Self-grooming is a common rodent behavior, and has an ordered cephalo-caudal progression from licking of the paws to head, body, genitals and tail. Acute stress is known to alter grooming activity levels and disrupt its patterning. Following 15-17 days of chronic social defeat stress, grooming behavior was analyzed in adult male C57BL/6J mice exhibiting either dominant or subordinate behavior. Our study showed that subordinate mice experience higher levels of anxiety and display disorganized patterning of their grooming behaviors, which emerges as a behavioral marker of chronic social stress. These findings indicate that chronic social stress modulates grooming behavior in mice, thus illustrating the importance of grooming phenotypes for neurobehavioral stress research.