The Developing Utility of Zebrafish Models for Cognitive Enhancers Research

Cognitive impairment is a common symptom in multiple brain disorders, including Alzheimer’s disease (AD), Huntingdon’s disease, autism and schizophrenia [ 1 - 4 ]. With the growing global elderly population, and cognitive decline observed with age, there is an urgent need for effective treatments of age- and neurodegenerative-related cognitive deficits [ 5 ]. With the limited spectrum of therapies currently available, and the general lack of model systems that fully recapitulate pathogenesis, predictive and high-throughput animal models become particularly important in the field of cognitive research [ 6 , 7 ]. While rodents have traditionally been used to study cognitive phenotypes [ 8 - 12 ], expanding the range of experimental domains and animal models is recognized as an important strategy of translational neuroscience research [ 13 ]. Although modeling neurobehavioral and cognitive disorders has primarily employed rodents in tests of learning, memory and attention [ 8 , 14 - 16 ], non-mammalian vertebrates and invertebrates such as Drosophila melanogaster , Caenorhabditis elegans and several fish species, have emerged as “complementary models” for such research [ 8 ]. Due to their complex nervous system and elaborate behavioral repertoire, zebrafish ( Danio rerio ) are gaining popularity as an excellent intermediate model between in vitro and in-vivo mammalian models for drug screening [ 7 ]. Drug discovery has traditionally utilized systemic, target-based screening in purified proteins or cells as primary screens, with in vivo models as tertiary screens after more mechanistic cell assays. Although in vitro screens have been successful in identifying molecules affecting known mechanisms, there is still the need to identify modulators of complex in vivo phenotypes in the whole organism for less well understood pathways – especially those occurring in a physiological and/or pathophysiological context. From this point of view, zebrafish offer a significantly higher throughput in vivo screening for phenotypic endpoints compared to other animal models [ 7 , 17 ]. Furthermore, zebrafish possess robust cognitive abilities, including learning and memory. For example, both associative and non-associative learning has been extensively tested in zebrafish, including cue-based and spatial learning as well as long-term and short-term memory [ 18 - 22 ]. Habituation, extensively investigated in animal models as an evolutionarily conserved adaptive behavior relevant to cognition [ 22 - 25 ], is also being increasingly assessed in zebrafish, as researchers continue to decipher the complexity of fish behaviors [ 21 , 22 , 26 ]. Notably, the habituation response in zebrafish can be modulated through various pharmacological manipulations, and is either attenuated or abolished depending on the receptor class targeted by the drug [ 26 ]. Memory has also been well studied in zebrafish, with the animals demonstrating the ability to recall a spatial alternation task for up to 10 days after testing [ 27 ]. Importantly, zebrafish cognition is also subject to normal aging processes, as conditioned responses to spatial, visual and temporal cues (associated with changes in cognitive responses to emotionally positive and negative experiences) show reduced generalization of adaptive associations, increased stereotypic and reduced exploratory behavior, and altered temporal entrainment over the course of the zebrafish lifespan [ 28 , 29 ]. Since the genome and genetic pathways controlling signal transduction and development are highly conserved between zebrafish and humans [ 30 ], various molecular tools available render zebrafish particularly useful for determining the mechanisms of action of various classes of psychoactive drugs [ 31 ]. Zebrafish models of brain disorders underlying cognitive deficits can also be useful to screen potentially neuroprotective or nootropic compounds for drug development, revealing the mechanisms crucial for new therapeutic treatments [ 31 ]. Collectively, this suggests that zebrafish represent a useful model in cognitive neuroscience research [ 7 , 8 , 17 , 31 ]. With its small size and transparent larvae, the zebrafish also provides an ideal system to apply novel tools for imaging targeted subsets of neurons and manipulating their activity [ 32 ]. Their utility in the recently developed field of optogenetics is increasingly allowing researchers to link neural activity with behavior and detect biological events at the cellular level [ 33 - 37 ]. These novel approaches foster the ability to predict how changes in circuit function may lead to altered cognition and behavior relevant to neurobehavioral disorders [ 38 ]. Here we discuss recent advances in pharmacological, genetic and optogenetic approaches using zebrafish models to study cognitive impairments and search for novel cognitive enhancers.

The investigation of novel drug targets for treating cognitive impairments associated with neurological and psychiatric disorders is an important focus of neurobehavioral research. Many promising therapies are progressing through preclinical and clinical development, and offer the potential of improved treatment options for cognitive impairments in AD and schizophrenia. The use of both adult and larval zebrafish in large scale, high-throughput screens for various psychotropic drugs has been extensively validated [ 77 - 79 ]. Given the sensitivity of zebrafish behavior and cognition to various pharmacological manipulations [ 78 , 80 ], they represent a promising novel organism to study the effects of psychotropic compounds, such as those that may attenuate cognitive impairment in models of neurological disorders. The cognitive deficits observed in aging and neurodegenerative disorders (e.g., AD) have been linked to decreased cholinergic transmission [ 81 - 84 ]. While acetylcholinesterase (AChE) inhibitors are currently the first line treatment for AD [ 85 , 86 ], they have been less promising therapeutically, producing only modest improvements in cognitive function with undesirable side effects [ 87 - 89 ]. Thus, agents which increase the release of acetylcholine by direct activation of presynaptic nicotinic acetylcholine receptors (nAChRs) have been suggested as a potential alternative [ 90 ]. For example, nicotinic agonists (including nicotine) improve memory function in multiple cognitive assays in rodents and monkeys, while impairments have been frequently reported following treatment with nicotinic antagonists [ 91 - 94 ]. The pro-cognitive effects of nicotine have been established in several animal models [ 91 , 92 , 94 ], and the genetic linkage between nAChRs and several cognitive deficiencies has also been established clinically [ 95 , 96 ]. Overall, the importance of nAChR subtypes in modulating cognitive processes has been extensively validated, suggesting that activation of nAChR by selective nAChR ligands may be a viable approach to enhance cognitive performance. With the growing support for neuronal nAChRs as valid pharmacological targets, zebrafish have been recently used as a model system to study the role of specific nAChR subtypes [ 97 , 98 ]. Notably, the zebrafish genome contains the complete complement of human neural nAChR subunit genes. For 8 of the 12 human genes, exactly one zebrafish ortholog exists, while for the 4 remaining human genes, two zebrafish homologs have been identified [ 99 , 100 ]. Thus, while zebrafish have been used to assess the effects of nicotine on cognitive function [ 39 , 101 , 102 ], their role in studying nAChRs as putative drug targets is becoming particularly promising. While nAChRs have been implicated extensively in human and animal studies of attention, learning and memory, the α4ß2 and α7 nicotinic receptor subunits (comprising the majority of the nicotinic receptor subtypes within the brain) are heavily involved in cognitive functioning [ 103 ]. Notably, both nicotinic α7 and α4β2 receptors have recently been implicated in the nicotinic anxiolysis in zebrafish, and are significantly attenuated by either α7 or α4β2 antagonists [ 104 ]. Evidence suggests that nAChRs may contribute to cognitive function by acting in local circuits of the hippocampus and cerebral cortex. For example, in the rodent hippocampus, α7-containing nAChRs are located postsynaptically on inter-neurons (where they mediate fast cholinergic excitatory synaptic transmission) and presynaptically (where their activation increases intra-terminal Ca 2+ levels and facilitates glutamate release) [ 105 - 107 ]. The hippocampus receives afferent projections, and contains a wide distribution or α7 nAChR receptors, thereby rendering it an ideal circuit to study the effects of α7 in sensory gating . In particular, deficits in PPI in sensory gating have been shown in schizophrenic patients, and serve as a primary means of assessing higher-order processing known to be involved in cognitive functioning. [ 108 - 110 ]. Moreover, PPI is also capable of being modulated in rodents as a function of nAChR activation [ 111 - 113 ]. In line with this, modulation of PPI in the zebrafish startle response may also be possible, similar to the response observed following dopamine agonist administration [ 70 ]. Similar to the effects demonstrated in humans [ 114 - 116 ] and rodents [ 117 - 119 ], antipsychotic drugs suppress PPI disruptions evoked in the zebrafish startle test [ 70 ]. However, whereas antipsychotic drugs are widely used for the treatment of psychotic symptoms in patients with schizophrenia, typical (first-generation) antipsychotics often lead to severe motor side effects due to the blockade of dopamine D 2 receptors [ 120 ]. Alternatively, atypical (second-generation) antipsychotics are increasingly used in the treatment of schizophrenia [ 121 ]. Atypical antipsychotics, although less potent in

In parallel with pharmacological and genomic modeling of cognitive impairment, mounting evidence shows the feasibility of detecting and tracking these diseases via disease-specific morphological signatures [ 143 ]. For example, clinical imaging studies have linked AD to brain atrophy [ 144 , 145 ] and Parkinson’s disease to decreased grey matter [ 146 , 147 ]. In line with this, the application of bioimaging techniques to several animal models and the development of digital brain atlases are revealing the bidirectional relationships between functional connectivity and disease pathology such as neurodegeneration [ 143 ]. Such connectome -based analyses in conjunction with digital atlas technology further increases our understanding of how cognitive processes emerge from their morphological substrates, and how these processes are affected when this structural substrate is disrupted [ 148 ]. With the growing utilization of zebrafish in neuroscience research, a population-based atlas of the zebrafish brain has recently been established, serving as a digital reference for anatomical localization comprising delineations based upon multiple magnetic resonance sequences and histological stains [ 143 ]. This publically available atlas provides important information on the spatial relationships of neuronal structures, as well as quantitative information and an accurate stereotaxic coordinate system. The combination of voxel-based comparisons and parametric comparisons for individual regions may also allow the effects of genetic and/or environmental manipulation on the anatomical phenotype to be assessed [ 143 ]. Recently, digital atlas data has also been further integrated with the visualization of gene expression data to allow researchers to identify spatiotemporal and quantitative aspects of gene expression in different stages of zebrafish development [ 149 ]. The use of connectome -based analyses helps to comprehensively map the neural connections from the level of single neurons and synapses (microscale) to the level of anatomically distinct brain regions and inter-regional pathways (macroscale) [ 148 , 150 - 152 ]. While currently focused on the human brain, connectome -based approaches may be further extended to animal models of neuropathology [ 152 ]. For example, the use of Brainbow imaging techniques has successfully revealed the connectivity between different neural populations in mice and Drosophila [ 153 - 155 ]. With rapid advances in the fields of optical microscopy and fluorescent dyes, further incentive to use an optically accessible model such as the zebrafish is supported. As zebrafish have fewer neurons than mice, more extensive networks of connectivity between different brain regions may be determined, also reflecting other highly dynamic processes, such as axon growth and pruning, which may be used as potential biomarkers of cognitive functions [ 156 ]. With the advent of new genetically encoded optical tools, researchers have recently begun to directly manipulate neuronal firing, probe neuronal-circuit function and control behavior on milliseconds timescales. The emergence of optogenetics has enabled monitoring and control of neuronal activity with minimal perturbation and unprecedented spatiotemporal resolution [ 157 - 161 ]. Briefly, light-gated channels and pumps allow the activation and silencing of neurons, while fluorescent proteins further allow for the sensing of calcium or membrane potential. Importantly, such optogenetic tools provide significant opportunities for analyzing neural circuits, especially those involved in cognitive and behavioral disorders [ 158 , 161 , 162 ]. For example, circuit-level perturbations in the brain's electrical activity may underlie cognitive deficits in schizophrenia and autism – disorders hypothesized to arise from hyperexcitation within neural microcircuitry. However, direct investigation of cell- and circuit-level effects of changes in cellular excitation has been precluded, as cell-specific pharmacological agents are lacking, and homeostatic processes can occur downstream of synaptic and intrinsic excitability alterations. Emerging optogenetics techniques have recently begun to bridge this gap, having been applied to study altered cellular excitation as well as quantify the effects on information transmission network activity in freely moving animals [ 162 ]. Zebrafish offer an ideal system to apply optical techniques for imaging the nervous system, as well as genetically encoded tools for targeting subsets of neurons and manipulating their activity [ 32 ]. Notably, its small size allows all neurons from a defined circuit to be monitored at once under a laser scanning microscope. For example, photoconvertible proteins can define and image circuits as well as link zebrafish behaviors with neuronal development, such as in developing spinal circuitry controlling tail movements in larvae [ 163 ]. Optical reporters of neuronal activity have also been useful

The authors confirm that this article content has no conflicts of interest.