The economic stakes could hardly be higher. Developing an innovative drug takes on average more than ten years, roughly eight of which are consumed by clinical trials, and the out-of-pocket cost per approved drug is estimated at approximately 173 million dollars, rising to about 879 million once failures and capital costs are included. Between 2014 and 2023, the likelihood that a candidate entering Phase I would ultimately win FDA approval was a mere 6.7 percent, with Phase II acting as the deadliest filter, advancing only 28 percent of the molecules it receives. A comprehensive review of AstraZeneca’s small-molecule projects from 2005 to 2010 showed that neural toxicity in the central nervous system accounts for nearly one quarter of failures across the entire discovery process, yet only a small fraction of these problems becomes evident during preclinical Good Laboratory Practice toxicity testing.

The reason most functional neural side effects slip through is that they do not leave the structural fingerprints that standard animal models are designed to catch. Disturbances of central nervous system function such as dizziness, anxiety, seizures or depression are far harder to predict than structural damage visible in histological sections. The Northeastern team’s Neuro-Behavioral Assay, originally developed to evaluate anesthetics, attacks this gap from a different direction by exploiting a biological quirk of the brown ghost knifefish, Apteronotus macrostomus of the Apteronotus leptorhynchus species group. This teleost generates a continuous, extraordinarily regular train of electric organ discharges, with amplitudes below one volt and frequencies approaching one kilohertz, and every single discharge is driven one-to-one by command spikes from an endogenous oscillator in the medulla oblongata called the pacemaker nucleus.

That tight coupling is what makes the assay so powerful: the frequency of the electric organ discharge is identical to the frequency of the neural oscillations of the pacemaker nucleus, so the fish’s electric behavior serves as a real-time, non-invasive proxy of activity in a specific brainstem structure. Electrodes built into an opaque PVC shelter tube record the signal continuously, which is then digitized at 50 kilohertz and parsed by automated procedures. Because the discharge frequency is extremely stable at constant temperature, with coefficients of variation in this study ranging from only 0.074 to 0.303 percent, even small drug-induced signals stand out clearly against the baseline. Two further readouts are extracted from the same recording: the rate of chirps, transient amplitude-frequency modulations driven by diencephalic input that probe higher brain function, and a locomotor activity index derived from amplitude changes as the fish moves relative to the electrodes.

The fourth and newest readout emerged from this study. After a 15-minute immersion in a 0.002 percent weight-per-volume solution of dextromethorphan, fish began quivering their anal and pectoral fins within two to six minutes, followed by jerking motions and belly-up postures, and then the most conspicuous effect: complete rotations about the longitudinal axis in the transverse plane, including full 360-degree turns that the researchers named Full Transverse Plane Rotations. These rotations, essentially absent in untreated and vehicle-treated fish, erupted in bouts and reached staggering rates of up to 165 per five minutes. Scoring was performed by an investigator blinded to treatment condition, and in five of the six fish the increase over baseline was highly significant, with rates persisting for hours and, in some individuals, for at least two days.

The dose-response data cemented the finding. Across four concentrations including a water-only control, a Spearman correlation revealed a significant moderate negative relationship between dextromethorphan concentration and electric organ discharge frequency, with all six fish showing frequency decreases at the highest dose ranging from 1.3 to 7.1 hertz against a mean control change of just 0.4 hertz. Chirp rate, which rose three- to eighteen-fold above baseline in three of the six fish, showed a strong positive correlation with concentration, and the net rotation rate correlated even more strongly. Notably, the chirp response appeared in only about half the animals, echoing a bimodal pattern previously seen with the anesthetics tricaine methanesulfonate and eugenol, a variability the researchers suggest may reflect the genomic and phenotypic diversity of the wild-type fish they deliberately use, a factor gaining recognition in drug discovery more broadly.

Crucially, the pattern of readouts produced by dextromethorphan differs from the signatures of every compound tested before. The anesthetics triggered dramatic locomotor suppression and discharge frequency drops exceeding 100 and reaching nearly 300 hertz, while diphenhydramine produced a more modest 40-to-100 hertz reduction without chirp or locomotor changes, and none of these drugs ever induced rotations. Dextromethorphan, by contrast, produced its characteristic trio of mild frequency decline, elevated chirping and vigorous rotational behavior, and its effects lingered far longer than the few-hour recovery typical of the other drugs, in one fish still clearly elevated on the third day after exposure.

Because the electric readouts are controlled exclusively by the pacemaker nucleus and its upstream inputs, any compound that alters them has demonstrably crossed the blood-brain barrier, which in ray-finned bony fish is built from the same tight-junctioned brain microvascular endothelial cells as in mammals and relies on conserved molecules such as Claudin-5. The authors therefore hypothesize that readout changes predict blood-brain barrier permeability in humans with reasonable likelihood, a capability current in silico and in vitro technologies have achieved only with limited success. The well-mapped circuitry of the pacemaker system also opens the door to target deconvolution: roughly 110 cells, about 90 pacemaker cells and 20 relay cells, generate the oscillation, and transient frequency changes are mediated by NMDA receptors while chirp-like modulations depend on AMPA receptors at the relay cells. The team proposes that dextromethorphan, a known NMDA receptor blocker, lowers discharge frequency by dampening excitatory drive at the relay cells, while a resulting glutamate surge stimulates AMPA receptors and drives chirping, a mechanism paralleling the drug’s antidepressant action that led to its repurposing for depression.

Translational parallels abound. Dextromethorphan is metabolized by the polymorphic enzyme CYP2D6, with 84 percent of studied volunteers classed as fast metabolizers and 9 percent as slow metabolizers whose drug half-life stretches from 2 to as long as 19 hours, and the researchers speculate that the fish homologue’s genetic variation might underlie the inter-individual differences observed in the assay. The prolonged behavioral impairment in the fish also contrasts strikingly with the popular assumption that intoxication wears off in three to six hours; the only controlled human study of supratherapeutic doses found psychomotor impairment, including a balance task, still measurable 360 minutes after administration. If the hypothesis holds that the diversity of affected readouts reflects a broad spectrum of side effects, as dextromethorphan’s documented profile of dizziness, restlessness, hallucinations and seizures suggests, the quadrimodal assay could let developers catch functional neural toxicity long before it detonates in a Phase II trial, quantify recovery on a continuous rather than binary scale, and even optimize molecules for brain penetration, all with a fish that broadcasts its brain activity through the water.

Subject of Research: A quadrimodal neuro-behavioral in vivo assay in weakly electric fish for detecting adverse central nervous system effects of drugs, validated with dextromethorphan

Article Title: Quadrimodal in vivo assay for detection of adverse neuro-behavioral effects: validation using dextromethorphan

Article References: Zupanc, G. K. H., Zodl, A. L., & Sabha, N. M. (2026). Quadrimodal in vivo assay for detection of adverse neuro-behavioral effects: validation using dextromethorphan. Frontiers in Drug Discovery, 6, Article 1917655. https://doi.org/10.3389/fddsv.2026.1917655

Image Credits: AI Generated

DOI: 10.3389/fddsv.2026.1917655

Keywords: dextromethorphan, neuro-behavioral assay, weakly electric fish, drug discovery, neurotoxicity, blood-brain barrier, pacemaker nucleus, electric organ discharge, NMDA receptors, AMPA receptors, preclinical screening, Apteronotus