Detection and characterization of functional (side-)effects of diphenhydramine in the central nervous system using a neuro-behavioral assay: proof of concept

Nearly 90% of drug candidates that enter clinical trials fail to receive approval from the U.S Food and Drug Administration. Approximately one-quarter of these failures are due to adverse effects in the CNS, most of which are caused by alterations in neural function. To address this issue, we employed a neuro-behavioral assay, originally designed for identification and characterization of the effects of general anesthetics on the neural oscillations of an endogenous brainstem nucleus in a fish model system. To establish proof of concept, we validated the applicability of this high-content in vivo assay by assessing possible neural (side-)effects of diphenhydramine, best known under its brand name Benadryl. In the neuro-behavioral assay, this antihistamine revealed a robust, reversible, dose-dependent neural effect, reflected by a pronounced drop in the oscillation frequency readout of the assay. This effect, presumably caused by use-dependent blocking of sodium channels, is consistent with secondary actions of diphenhydramine as a sedative and/or local anesthetic. The results of our study demonstrate the potential of the neuro-behavioral assay to predict functional (side-)effects of drug candidates in the central nervous system at earlier stages of drug development than possible with the currently available technologies.

Between 2010 and 2019, 38 new drugs were approved in the United States each year on average (Congressional Budget Office, 2021). During this period, the mean R&D expenditures per new drug totaled approximately $1 billion, including capital costs and expenditures on drugs that did not progress to the market. The latter adds tremendously to the costs of drug development, as, across all therapy areas, approximately 88% of drugs developed in the preclinical phase fail in clinical stages.

Among the major factors that contribute to the failure of drugs during the clinical phase is the difficulty to detect unwanted side effects of drug candidates in the central nervous system (CNS). Estimates suggest that this issue accounts for nearly one-quarter of failures across all stages of drug development (Walker et al., 2018). Most of these issues are detected only during clinical trials or post-drug marketing because they cause alterations in the function of neurons (manifested by sensory disturbances, cognitive impairment, and seizures, among others), rather than more easily detectable structural changes, evident as tissue damage.

Current technologies are limited in the ability to detect such adverse effects during early stages of drug development. They either examine the effect of a compound only on select neuroreceptors or ion channels (as done in in vitro electrophysiology), thereby failing to account for the complexity of interactions of neural systems that lead to unwanted side effects on the organism; or, they check primarily for general health deficits (as done by the widely used Irwin Test), without providing information about underlying neural dysfunctions.

To address this issue, we present a high-content in vivo assay, called the Neuro-Behavioral Assay (NBA), which evaluates possible neural (side-)effects of drugs in the CNS in a novel way. Based on the highly periodic electric organ discharge (EOD) of a weakly electric knifefish, the NBA utilizes this behavior as a proxy of the neural activity of an endogenous brainstem oscillator, the pacemaker nucleus. This is possible because the frequency of the EOD is identical to the oscillation frequency of the pacemaker nucleus, and changes in the oscillation pattern are reflected by corresponding changes in the EOD. The electric discharges can be recorded non-invasively through electrodes placed in the fish’s tank. Moreover, a single recording of the EOD provides, besides the EOD frequency, two additional readouts of the NBA—transient amplitude-frequency modulations of the EOD (referred to as ‘chirps’) and locomotor activity. Importantly, since changes in these readouts reflect an integrative neurophysiological response of several major brain systems induced by a neuroactive compound, they are a better indication of how the CNS reacts to a drug than possible by in vitro approaches.

The NBA was initially developed for the evaluation of neural effects of anesthetic drugs (Eske et al., 2023; Lehotzky et al., 2023; Ahmed and Zupanc, 2025; Zupanc and Ahmed, 2025). However, while many details of how anesthetics induce a state of anesthesia remain enigmatic, a more general property of anesthetic agents appears to be that they disconnect brain systems from the environment by modulating neural circuits involved in processing of sensory information and in controlling motor output (Kelz and Mashour, 2019). Since the pacemaker nucleus controls an important motor behavior of the fish—the EOD—we hypothesized that at least some of the compounds capable of modulating the function of motor systems would be detectable by the NBA.

We tested this hypothesis by examining the effect of diphenhydramine, an over-the-counter first-generation antihistamine that provides temporary relief of allergic reactions. Since diphenhydramine (widely known by the brand name Benadryl) has a long history spanning 8 decades, the adverse effects associated with its administration are well characterized. They include several effects thought to be caused by interference of the drug with functions in the CNS, such as drowsiness and dizziness, as well as seizures and cognitive decline in more severe cases (Agostini et al., 2001; Clark et al., 2025; Sicari et al., 2025). In line with such observations in human patients, the results of our study show that diphenhydramine induces a dose-dependent, reversible decrease in EOD frequency, thus providing proof of concept of the suitability of the NBA for detection of (side-)effects of some drugs on CNS function.

Animals

A total of 6 adult brown ghost electric knifefish (Apteronotus macrostomus) of the Apteronotus leptorhynchus species group (cf. de Santana and Vari, 2013) were used in this study. They originated from their natural habitat in Colombia and were imported by a tropical fish dealer (AliKhan Tropical Fish, Richmond Hill, NY, USA).

The body weight of these fish ranged from 6.9 g to 11.7 g (mean: 8.9 g), and the total length ranged from 130 mm to 175 mm (mean: 149 mm). Median baseline EOD frequencies, as measured in the experiments reported here, ranged from 692 Hz to 870 Hz, indicating that the sample included both females and males, as females tend to discharge at lower frequencies than males (Zupanc et al., 2014).

Several weeks before running an assay, the fish were transferred from a community tank to an isolation tank (50 cm × 30 cm × 30 cm), in the following referred to as ‘home tank.’ The latter was equipped with an aquarium thermostat heater and an air-driven corner filter. The fish was kept under a 12:12-h light-dark photoperiod at a conductivity of approximately 150–200 μS/cm, pH values of 7.7–7.9, and water temperatures of 26 °C–28 °C.

Readouts of the neuro-behavioral assay

The effect of diphenhydramine was assessed through the 3 readouts of the NBA—EOD frequency, chirp rate, and locomotor activity. Each of these readouts was based on the same recording of the fish’s EOD.

To produce the single EOD recording, the home tank of each fish was equipped with an opaque cylindrical PVC tube (length: 190 mm; inner diameter: 38 mm; outer diameter: 42 mm), which the fish, as nocturnal animals, utilize as daytime shelter (Figure 1). While running the assay, the open ends of the tube were closed with a coarse plastic mesh netting to prevent the fish from leaving the tube, thereby allowing uninterrupted high-quality recordings. Differential recording of the fish’s EOD was done through a pair of stainless-steel electrodes built into the tube. A strip of stainless steel placed near the tube served as a ground electrode. The recorded EOD signal was AC amplified (gain: 30×; low-pass filter: none; high-pass filter: 200 Hz) by a CED 1902 amplifier (Cambridge Electronic Design, Cambridge, England) and then digitized at a sampling rate of 50 kHz using a CED Micro 1401 mkII analog-to-digital converter (Cambridge Electronic Design), a Lenovo ThinkCentre V50s SFF desktop computer (equipped with an Intel Octa Core i7-10,700, 32 GB RAM, 512 GB NVMe +1 TB HDD), and the software program Spike 2 Version 5.21 (Cambridge Electronic Design).

The EOD frequency was calculated using the method by Lehotzky et al. (2023). Since the EOD frequency is temperature dependent, all frequency measurements were adjusted to a reference temperature of 26.0 °C, employing a Q 10 of 1.56 (Zupanc et al., 2003). Water temperature was measured every minute with a calibrated digital thermometer (Fisher Brand, Model 15–077–8, 11.705,843; Thermo Fisher Scientific, Waltham, Massachusetts, USA; accuracy ±0.05 °C). Chirps were detected using the method of Eske et al. (2023). Changes in locomotor activity, characterized by the Locomotor Activity Index (LAI), were assessed by analysis of amplitude modulations of the recorded EOD (Ahmed and Zupanc, 2025).

Experimental design

To assess the effect of diphenhydramine, each run of the NBA consisted of three parts (Figure 2). First, to establish baseline activity, the EOD of the fish was recorded in its home tank for 15 min. Then, the shelter tube with the fish was transferred to a separate, smaller tank (‘experimental tank; ’ 30 cm × 20 cm × 20 cm), where the recording continued during a 15-min exposure of the fish to diphenhydramine hydrochloride (Cat. No. A10136.18; ThermoScientific, Waltham, MA, USA) dissolved in water from the home tank (‘test run’); or to water from the home tank only (‘control run’). The handling of the shelter tube with the fish was identical under both conditions, and the temperatures of the water of the home tank and the experimental tank were very similar (±0.5 °C). Finally, to assess recovery from the treatment with diphenhydramine, the fish in its shelter tube was returned to the home tank, where recording of the EOD continued for either 1 h (‘short run’) or 5.5 h (‘long run’).

All fish survived the experiments and recovered, as indicated by return of each of the three behavioral readouts to baseline levels. This enabled us to use fish for multiple runs of the assay. However, no fish was subjected to more than one run on a given day. The reuse of fish enabled us to significantly reduce the number of animals needed for the present study.

Data analysis

The variability of sampled EOD frequency measurements was characterized by computing the coefficient of variation (CV) (Equation 1). It is, defined as

Statistical analysis of data was performed using IBM SPSS Statistics (version 29.0.1.1 (244)) (IBM Corporation).

Treatment of fish with diphenhydramine produced robust quantitative results in the EOD frequency readout of the NBA only. By contrast, we did not obtain any evidence that chirp rate and LAI were modulated by diphenhydramine within the range of concentrations tested (not shown).

Pilot experiments showed that diphenhydramine concentrations of 0.0003%–0.0047% result in reduction of EOD frequency only. At a concentration of 0.0067%, the decrease in EOD frequency was paralleled by marked amplitude fluctuations in 3 out of 6 fish, rendering the EOD signal unstable. It is likely that at higher concentrations of diphenhydramine the EOD signal would collapse, and the fish would potentially be killed. We observed amplitude collapse previously when we tested anesthetics at high concentrations in vivo (Lehotzky et al., 2023) and in vitro (Zupanc and Ahmed, 2025).

We interpret the findings in the present study as indication that lower concentrations of diphenhydramine lead to neuro-behavioral effects comparable to the relatively mild adverse neurological effects of the drug observed at therapeutic doses in humans, whereas higher concentrations cause rather severe disturbances of neural function, presumably mirroring the toxic and potentially lethal effects observed after diphenhydramine poisoning. Since the goal of our study was to focus on mild side effects, we restricted our testing to a maximum concentration of diphenhydramine in the immersion solution of 0.0067%.

Constancy of baseline EOD frequency

Detection of the changes in EOD frequency induced by diphenhydramine was greatly facilitated by a high signal-to-noise ratio of the NBA readout. This was possible due to robust decreases in the EOD frequency signal and minimal background, reflected by extremely low variability of the baseline EOD frequency (Figure 3). To characterize the degree of variability, we calculated the CV during the 15 min preceding the transfer of the fish from the home tank to the experimental tank. Since this baseline recording was identical for control experiments and any of the different types of experiments in which the fish were treated with diphenhydramine, we included all assay runs in this analysis. The mean CV values varied among individual fish but were rather constant for a given fish in different assay runs (Table 1). However, the CV in each assay run conducted in any of the 6 fish used in this study was extremely low, thus underscoring the usefulness of the baseline EOD frequency as a reference signal for assessing the effect of diphenhydramine on EOD frequency.

Effect of 0.0067% diphenhydramine

Immersion into a 0.0067% w/v diphenhydramine solution induced a rapid decrease in EOD frequency in each of the 6 fish examined within 1-3 min (Figure 3). This decline continued throughout the remaining time the fish were exposed to the compound in the experimental tank and, in some instances, even for 1–2 min after the fish’s return to the home tank. The lowest EOD frequency was 56 Hz (Fish MK-01), 37 Hz (Fish MK-02), 56 Hz (Fish MK-04), 47 Hz (Fish MK-06), 81 Hz (Fish MK-07), and 101 Hz (Fish MK-11) below the corresponding median baseline frequency.

Comparison of the EOD frequency values sampled during the last 5 min of the fish’s exposure to the diphenhydramine solution with the EOD frequency values sampled during the 15-min baseline recording showed, as expected from visual inspection of the time-frequency plots (Figure 3), highly significant differences in each of the 6 fish (p < 0.001, Mann-Whitney U-Test, 2-tailed).

Control runs

Control runs were conducted to examine whether handling of the fish during transfer from its home tank to the experimental tank containing only water from the fish’s home tank without diphenhydramine had any effect on the EOD frequency, compared to the baseline frequency. In 5 of 6 fish, the transfer induced a slight increase in median EOD frequency of 0.2–2.8 Hz (Figure 3). In the 6 th fish, the medians of the two frequencies were identical.

Comparison of the 180 EOD frequencies sampled during each of the two 15-min segments revealed significant differences in 4 of the 6 fish examined (Mann-Whitney U Test, p < 0.001, 2-tailed).

Dose-dependent effect of diphenhydramine on EOD frequency

To examine whether the magnitude of the decrease in EOD frequency induced by diphenhydramine was dose dependent, each of 3 fish (MK-04, MK-06, MK-07) was subjected to 6 test runs with concentrations (w/v) of the compound ranging from 0.0003% to 0.0067%, plus 1 control run (0% diphenhydramine). The maximum change in EOD frequency was used to assess the effect of the treatment. It was defined as the fish’s median EOD frequency during the last 1 min (n = 12 measurement) in the experimental tank minus the median baseline EOD frequency during the 15 min immediately preceding the fish’s transfer from its home tank to the experimental tank (n = 180 measurements). The results are shown in Figure 4. As expected from visual inspection of this plot, Spearman rank correlation analysis confirmed a strong dose-dependency (r S = −1.0 in Fish MK-04, -0.904 in Fish MK-06, and -0.929 in Fish MK-07; p < 0.05 in each fish).

In each of the 3 fish, immersion into diphenhydramine solution induced a highly significant (p < 0.001, Mann-Whitney U Test, 2-tailed) decrease in EOD frequency at concentrations equal or higher than 0.001%, as revealed by comparing the 60 EOD frequency measurements during the last 5 min in the experimental tank with the 180 EOD frequency measurements during the 15-min baseline recording. However, at the lowest concentration (0.0003%), statistically significant frequency decreases of 0.2 Hz and 0.9 Hz were induced only in Fish MK-04 and MK-06, respectively, whereas a frequency increase of 0.3 Hz was induced in Fish MK-07. The latter frequency difference was not significant at p < 0.001 (Mann-Whitney U Test, 2-tailed). These results indicate that the concentration of 0.0003% was close to the threshold concentration for detection in the NBA of an effect on EOD frequency caused by diphenhydramine.

We used the EOD frequency decreases induced by diphenhydramine in the concentration range of 0.0003% and 0.0067% for determining the Half Maximal Effective Concentration (EC50). As mentioned above, the frequency decrease evoked by the lowest concentration (0.0003%) is statistically not significantly different from baseline, whereas administration of the highest concentration (0.0067%) resulted in the largest decrease within what we assumed to be the therapeutic concentration range. These two endpoints yielded an EC50 of diphenhydramine in the immersion solution of 0.0067 % − 0.0003 % 2 = 0.0032%.

Recovery of EOD frequency

After transferring the fish from the experimental tank to its home tank, the EOD frequency gradually returned toward baseline frequency (Figure 3). To characterize the time course of recovery, we ran the assay in 4 fish according to the same protocol as described in Section 3.2 but extended the recording time after each fish’s return to its home tank to 5.5 h.

Time-frequency plots revealed two phases of recovery of the EOD frequency after the fish’s exposure to 0.0067% diphenhydramine: an initial phase characteristic of a steep rise in frequency, followed by a phase of rather slow further increase (Figure 5).

To quantify the degree of recovery, we first determined the time point at which the lowest EOD frequency induced by the fish’s exposure to the 0.0067% diphenhydramine solution had been reached. In each fish, the minimum frequency occurred toward the end of the 15 min in the experimental tank or shortly thereafter. Then, we determined how long it took the fish to (i) recover 90% of the difference between the minimum EOD frequency and the mean baseline EOD frequency and (ii) sustain this increased frequency thereafter. The resultant recovery times were 229 min (Fish MK-01), 106 min (Fish MK-02), 232 min (Fish MK-04), and 220 min Fish MK-11).

Notably, during the recovery phase, the time-frequency plot of each fish showed a notable degree of jitter, contrasting with the extremely low variability of the EOD frequency during the corresponding baseline recording. This jitter was most pronounced in Fish MK-11 (Figure 5), perhaps related to the finding that the CV values determined in this fish during baseline recording were higher than in any of the other 5 fish used in this study (Table 1).

Applicability of the NBA to anesthetics and other neuroactive drugs

Previous studies have demonstrated the power of the NBA as a high-content in-vivo assay for identification and characterization of (side-)effects of anesthetic drugs on CNS function (Eske et al., 2023; Lehotzky et al., 2023; Ahmed and Zupanc, 2025; Zupanc and Ahmed, 2025). Its advantages, compared to competitive technologies, include the possibility to extract three readouts from a single non-invasive behavioral recording (the EOD); to use one of these behavioral readouts (the EOD frequency) as a proxy of the neural activity (the oscillation frequency) of a brainstem system (the pacemaker nucleus); to achieve excellent separation from background noise (defined by baseline recording) of the EOD frequency signal after induction of anesthesia; to measure the phenotypic changes automatically (thus avoiding investigator’s bias) and on a continuous scale (as opposed to carrying out the measurements on a binary scale); to assess differential effects of drugs on different neural systems (as the normal EOD and modulations of the EOD are controlled by different brain systems); and to provide the perspective of target deconvolution, as the neural mechanisms controlling the generation of the oscillatory activity of the pacemaker nucleus and its modulations are well established (Metzner, 1999; Zupanc, 2002; Zupanc and Bullock, 2005; Zupanc, 2020).

In the present study, by testing diphenhydramine, we have provided proof of concept that the applicability of the NBA extends beyond anesthetics. Neural (side-)effects were revealed by one of the three readouts of the assay, the EOD frequency. No indication was obtained that diphenhydramine interferes with the neural functions underlying the other two readouts, chirp production and locomotor activity. The decrease in frequency was dose dependent. Administration of the drug at the highest concentration tested (0.0067%) induced frequency drops of up to 100 Hz within 15 min. These frequency decreases were less pronounced than those typically observed after subjecting fish to anesthesia, but clearly separated from background noise, statistically highly significant, and reproducible both within a given fish and among the entire sample of 6 individuals tested. The NBA also yielded detailed information about the persistence of the effect of diphenhydramine after exposure of the fish to the drug had been stopped, and about the time course of recovery of baseline neural activity.

Considerations on the concentrations used and their translational relevance

In the present study, we found concentrations of diphenhydramine between 0.001% and 0.0067% to be effective in inducing statistically significant decreases in EOD frequency without affecting the EOD amplitude. The onset of the frequency reduction within 1–3 min after the start of the fish’s 15-min exposure to diphenhydramine indicates that the drug was rapidly taken up from the immersion solution. It is, however, unknown what the corresponding drug concentrations were in the plasma and the brain.

A study in Asian Seabass (Lates calcarifer) showed that after immersion of the fish into a 140 μg/mL solution of the anesthetic tricaine methanesulfonate for 3 min, approximately half of this concentration was found in the plasma immediately after the exposure had stopped (Hsu et al., 2023). However, the plasma concentration decreased to about 10% of the initial plasma concentration after 5 min, to about 5% after 20 min, and to about 1% after 60 min. Thus, at these 3 time points only 5%, 2.5%, and 0.5% of the level of tricaine methanesulfonate present in the immersion solution was detectable in the plasma. Since recovery from tricaine methanesulfonate anesthesia occurs rapidly within a few minutes (Eske et al., 2023; Zupanc and Ahmed, 2025), it is likely that most of the initially taken-up anesthetic was excreted or metabolized at the times blood was sampled. Presumably, the fish’s gills play a major role in this uptake and excretion, as the gill epithelium is the site of many processes that are mediated by renal epithelia in terrestrial vertebrates (for review see Evans et al., 2005).

Although the dynamics of uptake and excretion of a molecule depend on a multitude of factors, it appears safe to assume that the level of diphenhydramine was much lower in the fish’s plasma than in the immersion solution, especially after the 15-min exposure of the fish to the drug had been stopped. In adult human subjects, a single 100 mg oral dose of diphenhydramine resulted in maximum concentrations of 81–159 ng/mL of the drug in the plasma 2–4 h after the administration (Glazko et al., 1974). In children and adolescents, with variable doses of 6–50 mg based on a weight-age-dosing schedule, maximum plasma concentrations were attained on average 1.5 h after the administration and ranged from 49–93 ng/mL (Gelotte et al., 2018). Whereas the maximum concentrations of diphenhydramine in the plasma found in these two studies are 2-3 orders of magnitude lower than the effective concentrations of the immersion solution used in the present investigation (10–67 μg/mL), given the findings by Hsu et al. (2023) it is possible that the plasma concentrations of diphenhydramine were much closer to those of human subjects after therapeutic dosing than to the concentration of the immersion solution. These considerations underscore the need to determine the concentrations of diphenhydramine in plasma and tissue samples from fish used in the NBA.

Neural mechanisms underlying behavioral readouts of the NBA

A major advantage of the NBA compared to other phenotypic in vivo assays is that the neural mechanisms controlling two of the three behavioral outputs—EOD frequency and chirp rate—are fairly well understood (Metzner, 1999; Zupanc, 2002; Zupanc and Bullock, 2005). (The central mechanism controlling the third behavioral output of the NBA—locomotor activity—is unknown.) The EOD is generated by synchronous depolarization of electrocytes—enormously enlarged axonal terminals of modified spinal motoneurons—that constitute the electric organ in the brown ghost electric knifefish (Zupanc and Bullock, 2005). These discharges are produced at high rates (up to 1,000 Hz) and exhibit extremely little variability, making the EOD the most stable periodic phenomenon known in neurobiology.

The discharges of the electric organ are driven by command spikes generated by the pacemaker nucleus—one volley of such output signals results in one EOD (Meyer, 1984). This one-to-one relationship conveys a unique competitive advantage to the NBA, enabling investigators to use the frequency of the EOD as a proxy of the frequency of the neural oscillations of the pacemaker nucleus.

The pacemaker nucleus is a well-defined brainstem structure whose oscillatory network consists of approximately 110 neurons — 90 pacemaker cells and 20 relay cells (Dye and Heiligenberg, 1987; Sîrbulescu et al., 2014; Zupanc et al., 2014; Ilieş and Zupanc, 2023). The pacemaker cells are connected via chemical and electrotonic synapses to each other and the relay cells (Elekes and Szabo, 1985; Dye, 1991). The relay cells convey the output signals of the pacemaker nucleus, via their axons, to the electromotoneurons.

Potential cellular and molecular targets of diphenhydramine in the pacemaker nucleus

Diphenhydramine is best known for its action as a competitive antagonist for histamine at histamine-1 receptors, and for its antagonistic activity at muscarinic cholinergic receptors (Sicari et al., 2025). In rats, histamine has been shown to facilitate high-frequency oscillations by potentiating the excitatory influence of brainstem fibers at the level of the basal forebrain (Dringenberg and Kuo, 2003). Other studies in rats have revealed muscarinic-receptor-mediated modulation of neural oscillations in the pedunculopontine nucleus in the brainstem (Ye et al., 2010; Kezunovic et al., 2013). Given these findings, it would be interesting to explore a possible association of these two transmitter systems with the pacemaker nucleus of the A. leptorhynchus species group. Yet, to our knowledge, no such study has ever been conducted. Our discussion here is therefore limited to a third well-established pharmacological action of diphenhydramine—blocking of sodium channels. Such a mechanism has been suggested previously to mediate the reduction of EOD frequency induced by two anesthetics, tricaine methanesulfonate (Eske et al., 2023; Zupanc and Ahmed, 2025) and eugenol (Lehotzky et al., 2023).

Electrophysiological studies in other systems have shown that the inhibition of neuronal sodium current by diphenhydramine is attributable to (i) selective binding of this agent to these channels in the open and inactivated state during high-frequency depolarizing stimuli and (ii) to slow dissociation of diphenhydramine from these channels between stimuli (Kim et al., 2000; Kuo et al., 2000). The use-dependent blocking of sodium channels appears to be particularly well suited as a key mechanism mediating the EOD frequency decrease in the pacemaker nucleus. As mentioned above (see Section 4.2), the pacemaker and relay cells fire at extremely high frequencies of up to 1 kHz, thus making sodium channels associated with these neurons particularly qualified as targets during inactivation. Similarly, the slow dissociation of diphenhydramine from sodium channels is consistent with the rather slow recovery of the EOD frequency, which, as shown in the present study, takes several hours after the exposure of the fish to the agent is stopped.

Implications of the neuro-behavioral changes in the NBA induced by diphenhydramine for functional (side-)effects in human patients

The demonstration of a robust effect of diphenhydramine on the neural function of the pacemaker nucleus is consistent with the well-established neural side-effects of this drug. Originally, in 1946, diphenhydramine was approved by the U.S Food and Drug Administration for management of allergic symptoms. However, the subsequently observed neural effects prompted a repurposing of diphenhydramine for several neurological applications (Khan and Tobias, 2025). Two of these applications are its use as a sedative and local anesthetic agent (Steffen et al., 1956; Carruthers et al., 1978; Green et al., 1994; Weiler et al., 2000; Pavlidakey et al., 2009; Xiao et al., 2023). Notably, a similar use-dependent block of sodium currents, as caused by diphenhydramine (see Section 4.3, above), is thought to mediate the effect of other local anesthetics (Ragsdale et al., 1994).

We hypothesize that the pronounced decrease in EOD frequency revealed by the NBA is caused by a similar molecular mechanism—use-dependent blocking of sodium channels—as are the effects of some general and/or local anesthetics. Differentiation of a compound’s potential for inducing general anesthesia versus sedation and/or local anesthesia can be achieved by evaluating its effectiveness on (i) the magnitude of the decrease in EOD frequency and (ii) reduction of locomotor activity. The general anesthetics tricaine methanesulfonate and eugenol induce EOD frequency decreases of up to several hundred hertz and a manifold reduction in locomotor activity in the NBA (Eske et al., 2023; Zupanc and Ahmed, 2025). By contrast, diphenhydramine, as shown in the present study, causes frequency decreases of up to 100 Hz and does not affect the locomotor-activity readout of the NBA, compared to controls.

If the link between EOD-frequency decrease in the NBA and sedative and/or local anesthetic (side-)effects will be further substantiated in future studies, then the NBA could be used as a powerful tool to predict such (side-)effects of drug candidates at early stages of drug discovery—much earlier than possible with the current technologies available. Such information could be used either for modification of the chemical structure of the candidate molecule to minimize the unwanted adverse effects, or for repurposing of this molecule for application in (other) specific neurological therapy areas.