A behavioral and molecular analysis of ketamine in zebrafish

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Ketamine exerts powerful anesthetic, psychotic, and antidepressant effects in both healthy volunteers and clinically depressed patients. Although ketamine targets particular glutamate receptors, there is a dearth of evidence for additional,
  A BEHAVIORAL AND MOLECULAR ANALYSIS OF KETAMINE INZEBRAFISH Sherry M. Zakhary 1 , Diana Ayubcha 1 , Farah Ansari 1 , Kiran Kamran 1 , Mehwish Karim 1 , Joerg R. Leheste 1 , Judith M. Horowitz 2 , and German Torres 1,* 1 Department of Neuroscience and Histology, New York College of Osteopathic Medicine of NewYork Institute of Technology, Old Westbury, New York, 11568, USA 2 Clinical Neuroscience Laboratory, Medaille College, Buffalo, New York 14214, USA Abstract Ketamine exerts powerful anesthetic, psychotic and anti-depressant effects in both healthyvolunteers and clinically-depressed patients. Although ketamine targets particular glutamatereceptors, there is a dearth of evidence for additional, alternative molecular substrates for thebehavioral actions of this NMDA receptor antagonist drug. Here, we provide behavioral andmolecular evidence for the actions of ketamine using a new vertebrate model for psychiatricdisorders: the zebrafish. Sub-anesthetic doses of ketamine produced a variety of abnormalbehaviors in zebrafish that were qualitatively analogous to those previously measured in humansand rodents treated with drugs that produce transient psychosis. In addition, we revealed that thetranscription factor Phox2b  is a molecular substrate for the actions of ketamine, particularly duringperiods of hypoxic stress. Finally, we also show that SIRT1 , a histone deacetylase widelyrecognized for its link to cell survival is also affected by hypoxia crises. These results establish arelevant assay system in which the effects of psychotomimetic drugs can rapidly be assessed, andprovide a plausible and novel neuronal mechanism through which ketamine affects critical sensorycircuits that monitor breathing behavior. Keywords Circling Behavior; Gill Movement; Hypoxia; Phox2b; Sirtuins INTRODUCTION Ketamine is a dissociative anesthetic capable of inducing post-operative hallucination,psychosis, and cognitive deficits in healthy individuals that bear an uncanny resemblance tothose observed in schizophrenic patients (Moghaddam, 2003). Yet, ketamine also appears toexert rapid and relatively sustained antidepressant-like effects at sub-anesthetic doses inpatients with major depression (Berman et al., 2000; Amiel and Mathew, 2007; Pilc et al., 2007; Pittenger et al., 2007). Because ketamine is an antagonist of the N-Methyl-D- Aspartate (NMDA) ion channel receptor, one of several subtypes of the glutamate receptorsystem (Leheste et al., 2008), it is thought that derangements in glutamate neurotransmissionmay contribute to the pathophysiology of both schizophrenia and major depressive disorder(Ross et al., 2006; Sanacora et al., 2007). * Corresponding Author:  German Torres, Ph.D. Department of Neuroscience and Histology NYCOM/NYIT PO Box 8000 OldWestbury, New York, 11568 USA Telephone: 516-686-3806 Fax: 516-686-3750 torresg@nyit.edu. NIH Public Access Author Manuscript Synapse . Author manuscript; available in PMC 2012 February 1. Published in final edited form as: Synapse  . 2011 February ; 65(2): 160167. doi:10.1002/syn.20830. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    Evidence for glutamate's role in psychiatric disorders also comes from animal models withvarying resemblance to the clinical features of a disease. For example, pharmacological ortransgenic manipulation of NMDA receptor signaling pathways in the rodent brain indicatethat ionotropic glutamate receptors have a profound influence on a wide-range of behaviorsthat are relevant to schizophrenia (Moghaddam, 2003; Engin et al., 2009). In this regard, there is a significant need to develop novel and better animal models for testing drugs totreat psychiatric disorders, including agents that target glutamate-based communication inspecific brain circuits. In the case of ketamine, we are using the freshwater zebrafish (  Daniorerio ) to study the neural networks that mediate the actions of psychotropic drugs and toidentify specific candidate genes that underlie the basis of ketamine-induced behaviors. Thezebrafish offers unrivaled opportunities for this line of investigation, primarily because of itsamenability to high throughput in vivo  drug screening (Guo, 2004; Flinn et al., 2008). This feature, together with a well-characterized stereotypical neuromuscular system and genetictractability offers a quick, easy and cost-effective way for modeling psychiatric disorders ina vertebrate organism. Thus, the present study describes a series of experiments in whichacute and chronic sub-anesthetic doses of ketamine were used to analyze species-specificbehaviors, and to identify behaviorally relevant molecular substrates for ketamine in theadult zebrafish. MATERIALS AND METHODS Animals Adult wild-type zebrafish (4-8 months) of mixed genders were obtained locally and handledin compliance with the NIH Guide for the Care and Use of Laboratory Animals and withapproval from the NYIT/NYCOM IACUC. Fish were kept at 28 °C in a recirculationaquaculture system equipped with carbon filtration, ultraviolet light sterilizers, and bio-filtration (Aquatic Habitats) under a 12 hr light:dark cycle (lights on 0700) and fed twicedaily with a commercial fish diet. All experiments were performed during the lights onperiod. All efforts were made to minimize animal stress and to reduce the number of fishused for the experiments detailed below. Ketamine Administration On test day, zebrafish were removed from their aquatic habitat and placed individually in250 ml glass-beakers (10 cm length × 8 cm width × 7 cm depth) containing temperate,recirculation aquaculture normoxic water. After a 10-min acclimation phase, ketamine(Vetalar-HCl, Amtech Phoenix Scientific, St Joseph, MO) was dissolved in the aquaculturewater and then after a 5-min waiting period species-specific behaviors (e.g., swimmingbehavior, gill movement) were recorded and videotaped for further behavioral analyses. Weconducted several pilot studies to determine optimal, sub-anesthetic doses of ketamine forproducing desired degrees of behavioral effects. The studies described herein have utilizedthis experience and knowledge base. First, we determined that a ketamine dose of 200 μ ldissolved in 100 ml of aquaculture normoxic water (0.2% solution) was the optimal, sub-anesthetic dose for our experimental purposes. Second, we determined that a solutionconcentration of 0.8% ketamine was a physiological anesthetic dose for this particularfreshwater animal as it produced a deep level of unconsciousness. Thus, the ketamine doseswere selected because they are sub-threshold (0.2%) or above threshold for an anestheticeffect in wild-type zebrafish. The above ketamine dose paradigm was instituted acutely andchronically for 5 consecutive days. To our knowledge, ketamine has not yet been applied tozebrafish for pharmacological studies. Zakhary et al.Page 2 Synapse . Author manuscript; available in PMC 2012 February 1. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    Behavioral Testing Procedures Behavioral activity (i.e., circling behavior) was monitored for 5-min and the number of complete, full (right or left) 360° circles were scored and videotaped following ketamine(experimental group; n = 20 fish) or no ketamine exposure (control group; n = 20 fish). Astress response test (i.e., hypoxic stress) was also conducted either acutely or chronically for5 consecutive days. In brief, after ketamine or no ketamine exposure, individual zebrafishwere removed from the aquaculture water for a 20-sec testing period during which time thenumber of gill movements (breaths) were recorded as well as the number of body pulses(“flops”). This particular stress response test was chosen because it provokes a ventilatorychemoreflex response in zebrafish. Thus, we established first a functional ventilatorychemoreflex response frequency in drug-naïve animals and then compared this baselineresponse frequency to that of ketamine-treated fish. After the hypoxic stress response test,animals were transferred to aquaculture fish chambers for 90-min and then sacrificed bydecapitation. Subsequently, their brains were excised from the skulls and processed forquantitative polymerase chain reaction (QPCR) procedures. Gene Expression Analysis by QPCR Procedures Zebrafish brain tissue was homogenized and RNA extracted using RNeasy® Plus Mini Kit(Qiagen, Carlsbad, CA), and QIAshredder™ (Qiagen Carlsbad, CA). RT-PCR wasperformed using the cDNA made with Superscript® III First-Strand Synthesis System forRT-PCR (Invitrogen, Carlsbad, CA). Expression of Phox2b , SIRT1  and  Actin  genes wasdetermined by QPCR with Power SYBR® Green PCR Master Mix (Applied Biosystems,Warrington, UK). Gene-specific DNA primers were manufactured using Integrated DNATechnologies (Coralville, IA). The  phox2b  primer sequence was forward 5 ′ - ACA ATCCCA TCA GGA CGA CGT TTG -3 ′  and reverse 5 ′ - TTC AAG CCT CCG TGA TCG GTGAAA -3 ′ . The SIRT1  primer sequence was: forward 5 ′ - ACA GTT CCA GCC ATC TCCATG TCA -3 ′  and reverse 5 ′ - AAG ACC CGT GGC ACT GAA TGA TCT -3 ′ . The  β  -actin primer sequence was forward 5 ′ - CAG CCA TGT ACG TTG CTA TCC AGG -3 ′  andreverse 5 ′ - AGG TCC AGA CGC AGG ATG GCA TG -3 ′ . Data Analyses Behavioral data are reported as means ± SEM. Analyses of Variance (ANOVA) followed byMann-Whitney Rank Sum Tests were performed with the assumption of unequal variance totest for differences between group means. Statistically significant differences were definedas P   0.05. Relative gene expression was analyzed using 2 ΔΔ Ct methods (Livak andSchmittgen, 2001). RESULTS Antagonist drugs of the NMDA-type glutamate receptor, such as ketamine andphencyclidine (PCP), can induce psychosis, as well as some of the behavioral signscatalogued in non-medicated schizophrenia patients, including rotational (circle) behavior.In this study, a sub-anesthetic dose of ketamine (0.2%) also produced a consistent circlingbehavioral phenotype in zebrafish, with individual turning rates ranging from 35 to 50 fullbody turns per 5-min testing period (Figs. 1, 2). This aberrant behavioral phenotype, incontrast, was completely absent in control animals (P   0.01). Instead, spontaneousbehavioral activity in control zebrafish showed chamber exploration with oblique turns andhalf-turn rotations accompanied by high percentage frequencies of apparent exploratorytasks. The high degree of circling behavior and hyperactivity in ketamine-treated zebrafishpersisted for longer than the 5-min testing period and were still observed even after theanimals had been transferred to aquaculture fish chambers (i.e., less than 2 min). Further, theaforementioned locomotor activity syndrome was characterized by lateralized circling Zakhary et al.Page 3 Synapse . Author manuscript; available in PMC 2012 February 1. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    behavior (e.g., a right-preference population bias), postural asymmetry, and hyperactivity tosensory stimuli. These initial findings indicate that application of ketamine to zebrafishproduces an abnormal behavioral phenotype that resembles that of healthy volunteers usingketamine (Krystal et al., 1994;Newcomer et al., 1999;Lahti et al., 2001), and mouse models with varying resemblance to the clinical features of schizophrenia (Mohn et al., 1999;Torreset al, 2004;Torres et al., 2005). Further, the fact that zebrafish were especially sensitive tothe psychotomimetic effects of ketamine, suggest that ionotropic NMDA receptor sub-unitsin this vertebrate animal might be homologous in function to those occurring in mammalianbrains.The actions of ketamine and the ventilatory response to hypoxia are critically dependent onionotropic NMDA glutamate receptors (Ohtake et al., 2000; Turesson et al., 2006; Maeng and Zarate, 2007; Leheste et al., 2008). This commonality in molecular substrates suggeststhat ketamine exposure might affect the general ventilatory response to hypoxia in zebrafish.To test this possibility, we assessed gill movement and the stress response (i.e., body“flops”) to a 20-sec hypoxic challenge. In this context, most of the sensory receptorsresponsible for triggering an oxygen ventilatory chemoreflex response in fish are located togill filaments (Sundin and Nilsson, 2002). Under this experimental condition, we found thatindividual animals treated acutely with 0.2% ketamine exhibited a profound (P   0.01)reduction in both gill movement and stress response to hypoxia relative to control, drug-naive zebrafish (Fig. 2). In particular, we observed an almost complete lack of gillmovement in ketamine-treated zebrafish, indicating a significantly decreased respiratorydrive in these behaving animals. It should be noted that the 20-sec hypoxic challengeinstituted in this experiment did not precipitate any deaths as all ketamine-treated zebrafishsurvived the procedure (data not shown). Within this brief period of hypoxia, we also noteda significant decrease in behaving activities (e.g., rapid turns and body “flops”) in animalsexposed to 5-min of ketamine. This suggests that ketamine either reduces the acute hypoxicstress in zebrafish or suppresses the contribution of NMDA receptors to respiratory drivetransmission at respiratory motor neurons in intact animals. Regardless, the parallel decreasein gill movement and the stress response we observed during hypoxia crisis suggests thatthese events are profoundly affected by previous ketamine exposure.Next, we determined the chronic effects of ketamine on spontaneous behavioral activityusing the same experimental design used for the acute drug treatment. Thus, the primarydifference between this experiment and the previous one is that instead of acute ketamineexposure, individual zebrafish were now exposed to 5 consecutive days of 0.2% ketamine.Under this chronic drug paradigm, we observed that repeated administration of ketamine didnot cause tolerance or sensitization to specific drug effects (Fig. 3). In general, thepharmacological effects of chronic ketamine administration were almost identical to thosepreviously observed in the acute drug preparation. That is, ketamine produced a consistentcircling behavioral phenotype in zebrafish that was completely absent in control, drug-naiveanimals (P   0.01). As described in the previous acute experiment, the recorded locomotoractivity syndrome was also characterized by lateralized circling behavior (e.g., a right-preference population bias), postural asymmetry, and hyperactivity to sensory stimuli.Further, we observed that animals treated chronically with 0.2% ketamine exhibited aprofound (P   0.01) reduction in both gill movement and stress response to hypoxia relativeto control, drug-naive zebra fish (Fig. 3). In particular under this chronic drugadministration, we observed an almost complete lack of gill movement in ketamine-treatedzebrafish, indicating a significantly decreased respiratory drive in these behaving animals.We also noted a significant decrease in behavioral activities (e.g., rapid turns and body“flops”) in animals exposed to 5 consecutive days of ketamine (Fig. 3). However, none of these deficits (i.e., diminished breathing capacity and reduced ventilatory chemoreflexresponse) were recorded in control animals kept in aquaculture fish chambers. Thus Zakhary et al.Page 4 Synapse . Author manuscript; available in PMC 2012 February 1. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    regardless of temporal drug treatment, ketamine not only seems to affect spontaneousbehavioral and breathing activity but also acts very swiftly throughout single or multipledrug exposures. This is consistent with the clinical view that single intravenous ketamineinfusions rapidly produce optimal therapeutic effects in treatment-resistant major depression(Liebrenz et al., 2007).To identify novel molecular substrates for the actions of ketamine during hypoxia crisis, weperformed QPCR in brains of drug-naïve and ketamine-exposed zebrafish. We selectedgene-specific primers for Phox2b , a hindbrain transcription factor predominantly expressedin neurons controlling breathing behavior (Guyenet, 2008) and sirtuin 1 ( SIRT1 ), anoxidation-reduction (REDOX)-sensitive deacetylase that stimulates the activity of hypoxia-inducible factor 2 α  (HIF-2 α ) during oxygen scarcity (Dioum et al., 2009). Against thisbackground, acute administration of ketamine to zebrafish caused a significantly greaterreduction (P   0.01) in the expression of both Phox2b  and SIRT1  during hypoxia crisis thanthat seen in brains from control, drug-naïve animals (Fig. 4). More specifically, QPCRanalysis of zebrafish treated with ketamine and then exposed to hypoxia showed asignificant reduction in Phox2b  expression relative to normalized levels of  β  -actin  inzebrafish collected immediately from aquaculture fish chambers. This transcriptionalrepression was similar in magnitude to levels observed in brains collected from animalsexposed only to ketamine or hypoxia (i.e., ~41% vs. 44% and 54%, respectively; P   0.05).Thus, no synergistic effects were measured as the combined actions of ketamine andhypoxia failed to produce a greater diminution of Phox2b  transcription levels than the sumof ketamine or hypoxia alone. These data support a mechanism in which NMDA receptorinhibition accounts for the reduction of Phox2b  transcripts in the zebrafish brain. These dataalso advance the idea that Phox2b  is a novel molecular substrate target for ketamine.Our QPCR measurements also showed SIRT1  transcripts to be significantly (P   0.01)reduced in brains of animals exposed to both ketamine and hypoxic stress (Fig. 4). It isnoteworthy that zebrafish exposed only to ketamine showed a modest (i.e., 5%), but notstatistically significant reduction (P   0.05) in SIRT1  transcription levels relative tonormalized levels of  β  -actin  measured in drug-naïve animals. Hypoxia alone, in contrast,produced a profound and statistically significant reduction (P   0.01) in brain SIRT1 transcription levels, suggesting that during oxygen scarcity, substrates for SIRT1  are eitherdegraded or destabilized in order to maintain transcription of the deacetylase. Further, thesedata suggest that SIRT1  itself is negatively influenced by hypoxic stress. Thus, levels of  SIRT1  transcripts in the zebrafish brain are proportional to conditions of hypoxia but notketamine. A possible reason for the augmentation of SIRT1  transcription levels following0.2 % ketamine and hypoxic stress could be explained by the persistent increase of neuronalsuperoxide and other reactive oxygen species (ROS) generated by NMDA receptorantagonists (Behrens et al., 2007;de Oliveira et al., 2009). DISCUSSION Schizophrenia and major depression have, for a long time, been regarded as having verydifferent srcins because of their distinct clinical symptoms and modes of drug treatment.However, these disorders are closely related to each other because their etiologies are clearlyrelated to functional alterations in catecholamine and glutamate systems that can spreadfrom one system to another and are precipitated by environmental insults in genetically-prone individuals. Thus, common characteristics of schizophrenia and major depressionsuggest parallel modes of disease pathogenesis and parallel approaches to treatment. If thisis the case, then sensible models of certain aspects of the neurobiological underpinning of these psychiatric disorders should be useful in establishing high-throughput drug screeningfor novel molecule probes that modulate a specific psychiatric disease mechanism. Results Zakhary et al.Page 5 Synapse . Author manuscript; available in PMC 2012 February 1. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  
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