To date, most pharmacological studies of behavior have been relatively small scale, involving a few small molecules at most. Here, we describe the behavioral phenotypes induced by thousands of small molecules, all tested under the same carefully controlled conditions. This large—scale data set provides opportunities for 1) systematic neuroactive drug discovery in the context of the intact nervous system and 2) improving our understanding how chemicals affect the brain and behavior.
Large—scale behavioral screening provides an opportunity to discover novel neuroactive compounds for research and therapy. For example, the STR and MAG compounds described here are novel AChE inhibitors (STR—1, STR—2) or MAO inhibitors (MAG compounds) that could have therapeutic activity for treating memory loss and depression, respectively12,13
. It is worth noting that for each of these novel compounds, comparison with existing neuroactive drugs was central to predicting their mechanisms of action. In the case of the STR compounds, we observed that they caused behavioral phenotypes similar to those caused by known AChE inhibitors. In the case of the MAG compounds, the SEA algorithm relied upon structural similarities between the MAG compounds and existing MAOIs to predict mechanisms. Not surprisingly, mechanism prediction is easiest when phenotypic or structural similarity exists between novel and known compounds. However, not all behavior—modifying compounds will have structural or phenotypic similarity to well characterized molecules. In these cases, other target identification strategies will be necessary.
Future screens may be devised to identify compounds that suppress pharmacologically induced behavioral defects. These screens could be performed using the PMR assay or other behavioral assays. The feasibility of such an approach is supported by our observation that the PMR assay can detect the suppression of pharmacologically induced behavioral defects ranging from organophosphate—induced immobility to stimulant—induced hyperactivity (). Beyond pharmacologically induced phenotypes, it may be possible to generate genetic models of nervous system disorders and screen for small molecule modifiers of the associated behavioral defects. Recently developed technologies for targeted gene mutation in zebrafish14–16
are likely to aid such efforts.
Behavioral assays like the PMR may also be useful for screening chemicals for neurotoxicology or other undesirable behavioral effects. Testing drug candidates for neurotoxicity remains a significant challenge, and new regulations requiring the testing of thousands of compounds are expected to require millions of rodents and cost billions of dollars17
. Given these costs and the ethical implications of increased animal use, inexpensive, high—throughput means of testing for neurotoxicity are needed, especially those involving non—mammalian species. Of course, questions remain about the degree of conservation of nervous system drug effects in zebrafish and humans. Although we found that many drugs with psychotropic effects in humans also cause reproducible behavioral effects on the PMR, many other psychotropic drugs failed to produce any detectable change in the PMR screen. We do not yet know the degree to which these “false negatives” are due to screening dose, failure of absorption, or imperfect conservation between zebrafish and humans. Clearly, answering these questions will be an important step toward translating behavioral pharmacology findings from zebrafish to humans.
In addition to its potential applications for drug discovery, large—scale behavior—based chemical phenotyping provides a new perspective on the relationship between small molecules and behaviors. In the experiments described here, the principal behavior under study had not previously been characterized. Despite this, the systematic approach we employed has begun to provide some hints about how the PMR is regulated. The fact that adrenergic, dopaminergic, serotonergic, and other drug classes modify the PMR in distinct ways suggests that these neurotransmitter pathways all contribute to PMR regulation. In addition, the fact that specific molecules modulate individual features of the PMR (such as the response latency, the excitation magnitude, excitation duration, etc.) suggests that this poorly understood behavior is comprised of specific functional components under distinct mechanisms of control. This information provides testable hypotheses about the functional architecture of the behavior.
In summary, we have sought to combine the in vivo relevance of traditional, behavior—based phenotyping with the scale and automation of modern drug discovery. Compared to in vitro and cell based assays, systematic phenotyping in living vertebrate animals, provides a more holistic understanding of neuropharmacology, including those effects that are modulated by compound metabolism or caused by complex interactions with multiple biological pathways. Thus, behavioral barcoding in zebrafish could accelerate the pace of neuroactive drug discovery and improve our understanding of how chemicals affect the brain and behavior.