The LDB test has been used to determine the anxiolytic- and anxiogenic-like effects of drugs in rodents
20. It has several advantages, including being rapid and not demanding the training of animals, as well as the deprivation of water or food
33. The time spent in each compartment of the LDB apparatus reflects attraction or aversion, depending on whether it is illuminated or dark, respectively
34. Meanwhile, the decreased number of transitions between compartments indicates an anxiogenic phenotype
35. Moreover, prolongation of the latency to enter the dark compartment is an index of anxiolytic-like effect. It is suggested as one of the safest indicators of this effect in the LDB test
36.
In the current study, levamisole has been shown to enhance the latency to enter the dark compartment while interestingly attenuating the number of transitions in the LDB test. The time spent in the light or dark compartment remained unchanged. Taken together, these findings suggest a partial anxiolytic-like profile in the LDB rather than a uniformly anxiolytic pattern. The prolonged latency suggests a delay in the initial transition to the dark compartment, yet the reduced transition number may also reflect reduced exploratory behavior, altered risk-assessment behavior, or changes in shuttling behavior that are not exclusively attributable to anxiety.
Importantly, the effects of anxiolytic drugs on the number of transitions in the LDB test are controversial: some studies report an increase 37,38, while others show no change 39. Moreover, the transitions between compartments and locomotion decreased at the same time, suggesting that the drug may have a sedative effect and indicating a drug-induced depression of general activity 28. Conversely, the enhanced number of transitions without an increment in locomotion has been reported to reflect anxiolytic activity. When any drug raises the number of transitions and locomotion, it is considered a general motor stimulant and is accepted as a possible anxiolytic 33. However, this interpretation is not fully supported because there is no change in the total number of crossed lines in the OFT. The reason for this paradoxical situation may be due to several factors, including experimental design (i.e., pharmacological settings, route of administration, and dose regimen) 27.
The OFT is a standard neophobic paradigm of anxiety 27. The OFT is primarily useful for observing and assessing the efficacy of candidate anxiolytic drugs 40. In this paradigm, animals are allowed to explore an open field freely enclosed by walls 41. Anxiolytic drugs have been shown to enhance time spent in the central area and reduce thigmotaxis (the tendency to stay close to walls), thereby decreasing the time spent in the peripheral area and exhibiting reduced anxiety-like behavior 40. The number of entries into the central area is another measure of an anxiety state 27 and increases with anxiolytic-like effect 42. The total number of crossed lines and rearings is generally used as a marker of locomotor activity. However, they are also an index of exploration and anxiety. The high levels of these behaviors point to increased locomotion and exploration and indicate a decrease in anxiety levels 32. In the current study, the number of entries into the central area, the total number of crossed lines, the number of fecal pellets, the total number of rearings, and the duration of grooming did not change with levamisole treatment. However, the time spent in the central area increased, while the time spent in the peripheral area decreased in the levamisole group. This pattern suggests that levamisole may have altered spatial preference in the center rather than increasing exploratory transitions into the center. In other words, central avoidance may have been reduced without a clear increase in approach frequency. This dissociation between center time and center entries may reflect changes in risk-assessment or decision-making strategies, whereby animals enter the center with similar frequency but remain there longer. Overall, the OFT findings indicate a limited anxiolytic-like effect, reflected mainly in altered spatial preference rather than a generalized increase in exploratory activity.
Levamisole can inhibit MAO-A 6, a finding confirmed by our previous study 25. MAO-A is a dominant enzyme in the rat brain compared to MAO-B 43. MAO-A mainly degrades adrenaline, NA, and 5-HT 15. Consistent with this, it has been noted that MAO-A deficiency leads to increased 5-HT levels in the brains of mice 44. 5-HT is closely associated with various neuropsychiatric conditions, particularly anxiety disorders 45, although the relationship between 5-HT and anxiety is complex and not solely determined by absolute transmitter levels. Levamisole was reported to alter the metabolism of 5-HT in particular brain regions of rats 46. Accordingly, it is plausible that monoaminergic pathways—potentially involving serotonergic mechanisms and/or other MAO-A–related substrates—could contribute to the behavioral pattern observed here. However, this mechanistic interpretation remains speculative because MAO-A activity and expression, as well as regional monoamine levels, were not assessed in the present study.
Reviewing the literature, most studies on MAO-A inhibition have primarily assessed the acute effects of compounds with MAO-A inhibitory activity—encompassing MAOIs, antidepressants, and non-antidepressant agents—on OFT and LDB outcomes. In contrast, the present study evaluated chronic levamisole exposure, which is an important distinction when interpreting behavioral findings, as acute and chronic regimens can yield partially divergent profiles due to differences in pharmacodynamics and behavioral adaptation. Given the renewed clinical interest in levamisole in the context of COVID-19, clarifying its potential central and behavioral effects may be relevant for a more complete understanding of its overall pharmacological profile. In particular, when a drug gains broader clinical attention beyond its traditional indications, defining possible neurobehavioral effects can help contextualize its benefit–risk profile and guide future mechanistic studies. In two acute treatment studies, moclobemide, a reversible inhibitor of the MAO-A, increased the time spent in the light compartment but did not alter the number of transitions in the LDB test. In the OFT, it attenuated the number of rearings but not ambulation (i.e., horizontal activity) 47, suggesting a selective modulation of exploratory components rather than a generalized change in locomotion. Notably, in a subsequent acute treatment study, the same agent (at the same dose as in the previous study, 10 mg/kg), moclobemide again increased the time spent in the light compartment, but, in contrast to the earlier study, also increased the latency to enter the dark compartment and the number of transitions in the LDB test 36, indicating that even within the same compound and dose, LDB outcomes—particularly transitions—may vary across experimental conditions. Consistent with this variability, in another acute study, diphenyl diselenide, which has MAO-A inhibitory activity, also increased the time spent in the light compartment and the latency to enter the dark compartment in the LDB test, but the number of transitions between compartments did not change. Importantly, it also did not change the number of crossed lines and rearings in the OFT 48, supporting the notion that some MAO-A–related compounds can produce anxiolytic-like shifts in approach/avoidance measures without clear effects on general activity indices.
Extending these observations to another MAO-A–inhibitory compound, a recent study, J147, a derivative of curcumin, another MAO-A inhibitor, increased the time spent in the central area in different brain areas but did not alter the number of rearings in the OFT 49, again pointing to a partially selective profile in which center-related measures change in the absence of robust exploratory alterations.
Compared with these compounds, levamisole produced a partially overlapping but not identical behavioral profile. Unlike established MAO-A inhibitors, levamisole was not developed as a classical centrally acting MAOI and appears to have a broader pharmacological profile. Similar to several MAO-A inhibitors, levamisole increased latency to enter the dark compartment in the LDB test and increased time spent in the central area in the OFT, findings compatible with an anxiolytic-like effect. However, levamisole reduced the number of transitions in the LDB test, whereas previous studies with better-characterized MAO-A inhibitory agents generally reported either no change or an increase in this parameter. This difference suggests that levamisole does not fully reproduce the behavioral pattern of conventional MAO-A inhibitors. Moreover, most previous studies assessed acute treatment, whereas the present study evaluated chronic exposure. Accordingly, comparisons with established MAO-A inhibitors should be made cautiously, and the effects of levamisole may not be explained solely by a classical MAO-A inhibitory mechanism.
Regarding the studies investigating the effects of levamisole on anxiety-like behaviors, there are very few studies on locomotor activity, not directly about anxiety-like effects. Overall, the findings vary by dose and experimental context. In acute levamisole treatment at 1 mg/kg, locomotor activity was reported as unchanged in one study 5, whereas another study observed a transient increase 50. At 5 mg/kg, acute treatment increased locomotor activity, whereas at 10 mg/kg, no change was observed 5. In a protocol most comparable to the present study in terms of dose and route (2 mg/kg by gavage daily for 10 days), levamisole reduced total distance in the OFT 48, whereas a subchronic study reported no locomotor change, consistent with our findings 52. Taken together, these data suggest that levamisole-related locomotor outcomes may depend on dose, strain, and exposure duration, raising the possibility of a non-linear dose–response relationship and indicating that results obtained at 2 mg/kg may not necessarily generalize to other doses.
Several limitations of the present study should be considered when interpreting the findings. First, an a priori power analysis was not performed. Although the sample size (n=10 per group) was determined based on group sizes commonly used in previous experimental animal studies, the study may still have been underpowered to detect subtle between-group differences. Therefore, non-significant findings should be interpreted with caution. In addition, multiple behavioral outcomes were analyzed without formal correction for multiple comparisons. Given the nature of the study, this approach may be acceptable; however, the absence of such correction may increase the risk of type I error, particularly when interpreting borderline significant findings. Additionally, only a single dose of levamisole (2 mg/kg) was evaluated; therefore, the dose–response relationship and the pharmacodynamic profile of levamisole with respect to anxiety-like behaviors could not be determined. Third, the behavioral results across LDB and OFT were not uniformly convergent, indicating a mixed profile that should be interpreted cautiously. Moreover, because the OFT was conducted on the same day immediately after the LDB test, potential carryover effects (e.g., stress, habituation, or learning-related effects from the first test) cannot be ruled out. Finally, mechanistic endpoints were not assessed; brain MAO-A activity/expression, regional brain monoamine levels, and peripheral or central biochemical measures were not evaluated. Future studies integrating behavioral testing with molecular and neurochemical assessments in relevant brain regions are warranted to elucidate the underlying pathways.
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