Learning and memory in zebrafish (Danio rerio).

Learning and memory are defining features of our own species inherently important to our daily lives and to who we are. Without our memories we cease to exist as a person. Without our ability to learn individuals and collectively our society would cease to function. Diseases of the mind still remain incurable. The interest in understanding of the mechanisms of learning and memory is thus well founded. Given the complexity of such mechanisms, concerted efforts have been made to study them under controlled laboratory conditions, ie, with laboratory model organisms. The zebrafish, although new in this field, is one such model organism. The rapidly developing forward- and reverse genetic methods designed for the zebrafish and the increasing use of pharmacological tools along with numerous neurobiology techniques make this species perhaps the best model for the analysis of the mechanisms of complex central nervous system characteristics. The fact that it is an evolutionarily ancient and simpler vertebrate, but at the same time it possesses numerous conserved features across multiple levels of biological organization makes this species an excellent tool for the analysis of the mechanisms of learning and memory. The bottleneck lies in our understanding of its cognitive and mnemonic features, the topic of this chapter. The current paper builds on a chapter published in the previous edition and continues to focus on associative learning, but now it extends the discussion to other forms of learning and to recent discoveries on memory-related features and findings obtained both in adults and larval zebrafish.

The Zebrafish, a Novel Model Organism for Screening Compounds Affecting Acute and Chronic Ethanol-Induced Effects.

Alcohol addiction is a major unmet medical and economic issue for which very few efficacious pharmacological treatment options are currently available. The development and identification of new compounds and drugs to treat alcohol addiction is hampered by the high costs and low amenability of traditional laboratory rodents to high-throughput behavioral screens. The zebrafish represents an excellent compromise between systems complexity and practical simplicity by overcoming many limitations inherent in these rodent models. In this chapter, we review current advances in the behavioral and neurochemical characterization of ethanol-induced changes in zebrafish. We also discuss the basic principles and methods of and the most recent advances in using paradigms with which one can screen for compounds altering acute and chronic ethanol-induced effects in zebrafish.

Molecular psychiatry of zebrafish.

Due to their well-characterized neural development and high genetic homology to mammals, zebrafish (Danio rerio) have emerged as a powerful model organism in the field of biological psychiatry. Here, we discuss the molecular psychiatry of zebrafish, and its implications for translational neuroscience research and modeling central nervous system (CNS) disorders. In particular, we outline recent genetic and technological developments allowing for in vivo examinations, high-throughput screening and whole-brain analyses in larval and adult zebrafish. We also summarize the application of these molecular techniques to the understanding of neuropsychiatric disease, outlining the potential of zebrafish for modeling complex brain disorders, including attention-deficit/hyperactivity disorder (ADHD), aggression, post-traumatic stress and substance abuse. Critically evaluating the advantages and limitations of larval and adult fish tests, we suggest that zebrafish models become a rapidly emerging new field in modern molecular psychiatry research.

Strain dependent gene expression and neurochemical levels in the brain of zebrafish: focus on a few alcohol related targets.

The zebrafish is becoming increasingly popular in behavior genetics because it may allow one to conduct large scale mutation and drug screens facilitating the discovery of mechanisms of complex traits. Strain differences in adult zebrafish behavior have already been reported, which may have important implications in neurobehavioral genetics. For example, we have found the AB and SF strains to differ in their behavioral responses to both acute and chronic alcohol exposure. In the current study, we further characterize these strains using semi-quantitative RT-PCR to measure the expression of ten selected genes and HPLC to measure the levels of nine neurochemicals. We chose the target genes and neurochemicals based upon their potential involvement in alcohol and other drugs of abuse related mechanisms. We quantified the expression of the genes encoding D1-R, D2a-R, D4a-R dopamine receptors, GABA(A)-R, GABA(B)-R1, GAD1, MAO, NMDA-R (NR2D subunit), 5HT-R1bd and SLC6 a4a. We found the gene encoding D1 dopamine receptor over-expressed and the genes encoding GABA(B1) receptor and solute family carrier protein 6 (SLC6) 4a under-expressed in SF compared to AB. We also found the level of all (dopamine, DOPAC, Serotonin, GABA, Glutamate, Glycine, Aspartate, Taurine) but one (5HIAA) neurochemicals tested decreased in SF as compared to AB. These results, combined with previously identified behavioral differences between the AB and SF strains, demonstrate the importance of strain characterization in zebrafish. They now also allow formulation of working hypotheses about possible mechanisms underlying the differential effects of acute and chronic alcohol treatment on these two zebrafish strains.

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.

EphA/ephrin-A interactions regulate epileptogenesis and activity-dependent axonal sprouting in adult rats.

The Eph family of tyrosine kinase receptors and their ligands, ephrins, are distributed in gradients and serve as molecular guidance cues for axonal patterning during neuronal development. Most of these molecules are also expressed in mature brain. Thus, we examine here the potential roles of such molecules in plasticity and activity-dependent mossy fiber sprouting of adult CNS. We show that the ligand ephrin-A3 and the receptor EphA5 are expressed in complementary gradients in the adult rat mossy fiber system. Using the kindling model, we demonstrate that exogenous immunoadhesins that affect the interaction of endogenous EphA receptors and ephrin-A ligands modulate the development of kindling, one type of long-term plasticity, in mature rat brain. These immunoadhesins, combined with epileptogenic stimulations, alter both the extent and the pattern of collateral axonal sprouting in the mossy fiber pathway. Our results suggest that EphA receptors and ephrin-A ligands modify neuronal plasticity and may serve as spatial cues that modulate the development and pattern of activation-dependent axonal growth in adult CNS.

Memory enhancement: the progress and our fears.

In a recent article Rose (2002) raises numerous crucial issues with regard to the research into and the use of cognition or memory enhancing agents. Although development of 'smart' drugs is in its infancy, his paper delineates some issues society may have to face when these drugs arrive. Questions about the development of such drugs may be interesting to several readers of Genes Brain and Behavior given the wealth of information expected to be gained on brain function from studies using genetic approaches including mutagenesis, transgenic techniques and genomics in general. Besides the scientific questions, several ethical issues may need to be addressed that are of interest to us all. Rose (2002) discusses some of these questions, but perhaps presents a too negative view on the problems, especially with regard to the present and future of memory research. This paper is intended to focus mainly on the scientific questions and argues that our fear of complex ethical problems should not make us throw the baby (i.e., our research and discoveries) out with the bath water.

Performance deficits of mGluR8 knockout mice in learning tasks: the effects of null mutation and the background genotype.

mGluR8 is a G-protein coupled metabotropic glutamate receptor expressed in the mammalian brain. Members of the mGluR family have been shown to be modulators of neural plasticity and learning and memory. Here we analyze the consequences of a null mutation at the mGluR8 gene locus generated using homologous recombination in embryonic stem cells by comparing the learning performance of the mutants with that of wild type controls in the Morris water maze (MWM) and the context and cue dependent fear conditioning (CFC). Our results revealed robust performance deficits associated with the genetic background, the ICR outbred strain, in both mGluR8 null mutant and the wild type control mice. Mice of this strain origin suffered from impaired vision as compared to CD1 or C57BL/6 mice, a significant impediment in MWM, a visuo-spatial learning task. The CFC task, being less dependent on visual cues, allowed us to reveal subtle performance deficits in the mGluR8 mutants: novelty induced hyperactivity and temporally delayed and blunted responding to shocks and temporally delayed responding to contextual stimuli were detected. The role of mGluR8 as a presynaptic autoreceptor and its contribution to cognitive processes are hypothesized and the utility of gene targeting as compared to pharmacological methods is discussed.

Behavioural and physiological characterization of inbred mouse strains: prospects for elucidating the molecular mechanisms of mammalian learning and memory.

With the advent of recombinant DNA methodology, it has become possible to dissect the molecular mechanisms of complex traits, including brain function and behaviour. The increasing amount of available information on the genomes of mammalian organisms, including our own, has facilitated this research. The present review focuses on a somewhat neglected area of genetics, one that involves the study of inbred mouse strains. It is argued that the use of inbred mice is complementary to transgenic approaches in the analysis of molecular mechanisms of complex traits. Whereas transgenic technology allows one to manipulate a single gene and investigate the in vivo effects of highly specific, artificially induced mutations, the study of inbred mouse strains should shed light on the roles of naturally occurring allelic variants in brain function and behaviour. Systematic characterization of the behavioural, electrophysiological, neurochemical, and neuroanatomical properties of a large number of inbred strains is required to elucidate mechanisms of mammalian brain function and behaviour. In essence, a 'mouse phenome' project is needed, entailing the construction of databases to investigate possible causal relationships amongst the phenotypical characteristics. This review focuses on electrophysiological and behavioural characterization of mouse strains. Nevertheless, it is emphasized that the full potential of the analysis of inbred mouse strains may be attained if techniques of numerous disciplines, including gene expression profiling, biochemical analysis, and quantitative trait loci (QTL) mapping, to name but a few, are also included.

Gene targeting: technical confounds and potential solutions in behavioral brain research.

Gene targeting allows one to create null mutations in mice and to analyze how the mutant organism responds to the lack of a single gene product. This has facilitated the molecular dissection of such complex characteristics as mammalian brain function and behavior, including learning, memory, aggression, and maternal behavior to mention a few. However, the interpretation of the phenotypical changes that arise in null mutant mice has been questioned. The possibility that genes other than the targeted one may contribute to phenotypical alterations has been raised and the importance of compensatory mechanisms has been brought to attention. This review focuses on recent advances in the literature that illustrate the caveats associated with gene targeting and also presents an overview of potential solutions for the discussed problems.

Behavioral tests of hippocampal function: simple paradigms complex problems.

Behavioral tests have become important tools for the analysis of functional effects of induced mutations in transgenic mice. However, depending on the type of mutation and several experimental parameters, false positive or negative findings may be obtained. Given the fact that molecular neurobiologists now make increasing use of behavioral paradigms in their research, it is imperative to revisit such problems. In this review three tests, T-maze spontaneous alternation task (T-CAT), Context dependent fear conditioning (CDFC), and Morris water maze (MWM) sensitive to hippocampal function, serve as illustrative examples for the potential problems. Spontaneous alternation tests are sometimes flawed because the handling procedure makes the test dependent on fear rather than exploratory behavior leading to altered alternation rates independent of hippocampal function. CDFC can provide misleading results because the context test, assumed to be a configural task dependent on the hippocampus, may have a significant elemental, i.e. cued, component. MWM may pose problems if its visible platform task is disproportionately easier for the subjects to solve than the hidden platform task, if the order of administration of visible and hidden platform tasks is not counterbalanced, or if inappropriate parameters are measured. Without attempting to be exhaustive, this review discusses such experimental problems and gives examples on how to avoid them.

Impaired water maze learning performance without altered dopaminergic function in mice heterozygous for the GDNF mutation.

Exogenous glial cell line-derived neurotrophic factor (GDNF) exhibits potent survival-promoting effects on dopaminergic neurons of the nigrostriatal pathway that is implicated in Parkinson's disease and also protects neurons in forebrain ischemia of animal models. However, a role for endogenous GDNF in brain function has not been established. Although mice homozygous for a targeted deletion of the GDNF gene have been generated, these mice die within hours of birth because of deficits in kidney morphogenesis, and, thus, the effect of the absence of GDNF on brain function could not be studied. Herein, we sought to determine whether adult mice, heterozygous for a GDNF mutation on two different genetic backgrounds, demonstrate alterations in the nigrostriatal dopaminergic system or in cognitive function. While both neurochemical and behavioural measures suggested that reduction of GDNF gene expression in the mutant mice does not alter the nigrostriatal dopaminergic system, it led to a significant and selective impairment of performance in the spatial version of the Morris water maze. A standard panel of blood chemistry tests and basic pathological analyses did not reveal alterations in the mutants that could account for the observed performance deficit. These results suggest that endogenous GDNF may not be critical for the development and functioning of the nigrostriatal dopaminergic system but it plays an important role in cognitive abilities.

Eph receptors and neural plasticity.

Eph receptor tyrosine kinases are largely known for their involvement in brain development but, as some of these receptor tyrosine kinases are also expressed in adults, their possible role in the mature nervous system has begun to be explored. Evidence for the involvement of Eph receptors in synaptic plasticity, learning and memory is only emerging and needs corroboration. However, it is likely that the actions of Eph kinases in the adult brain will attract significant attention and become a fertile research area, as occurred in the case of the neurotrophins.

Protein targeting: altering receptor kinase function in the brain.

Gene targeting has proved to be one of the most powerful techniques with which one can investigate molecular mechanisms that underlie complex phenomena such as learning and memory. Despite its popularity, however, concerns have been raised about this technique and alternative approaches have been sought. One such approach is protein targeting, which is based on the application of immunoadhesins, genetically engineered fusion proteins that exhibit functionally relevant target specificity. These immunoadhesins modulate the activity of not only a single receptor but of all receptors with homologous binding sites, which thereby eliminates the possibility of compensation by sister receptors. Furthermore, immunoadhesins can be used not only to impair but also to improve receptor function in the brain. Initial studies using immunoadhesins suggest that protein targeting might be a useful approach for analyzing the molecular mechanisms of brain function and behavior.

Heregulin, but not ErbB2 or ErbB3, heterozygous mutant mice exhibit hyperactivity in multiple behavioral tasks.

Genetic redundancy is a problem in gene targeting studies because functionally relevant sister proteins can compensate for the lack of protein product of a targeted gene. A molecular system is chosen in which it is hoped to demonstrate both the lack and presence of compensation after disruption of particular single genes. Mammals may not be able to compensate for the lack of heregulin, a single ligand for multiple ErbB receptors, however, compensation is expected when a single ErbB receptor is knocked out. To investigate this the heregulin-1, ErbB2, or ErbB3 locus was disrupted in a targeted manner and mice heterozygous for the mutation were analyzed. Heregulin and its receptors were shown to be involved in embryonic brain development and, more recently, in plastic changes associated with adult brain function in rodents. Although they have never been shown to play roles in mammalian behavior, it was decided to characterize the mice behaviorally using a battery of simple tests. Heregulin mutant mice exhibited elevated activity levels in the open field, showed improved rotorod performance, and finished T-maze spontaneous alternation task faster compared to control wild type littermates, findings that suggest a consistent hyperactivity across tests. ErbB2 and ErbB3 mutant mice, whose strain origin was identical to that of heregulin mutants, showed no sign of the behavioral alterations. It is suggested that the abnormalities seen in heregulin mutant mice are due to mutation at that locus and the lack of alterations seen in ErbB2 and ErbB3 mutant mice is the result of compensation by unaltered sister receptors.