Nocturnality is a behavioral trait characterized by the allocation of increased awareness and general activity (foraging, social interactions, territory patrolling) in a time window synchronized with the dark phase of the photoperiod, the night. This synchronization reflects entrainment of endogenous circadian rhythms to environmental daily cycles and mediates the colonization of a specific temporal niche. Freshwater South American weakly electric fish are nocturnal animals. Nocturnality has been explored in several species of this group, with behavioral approaches that focus on locomotor activity, electric behavior and electrocommunication. Early experiments showed a nocturnal increase in locomotor activity and exploratory behavior (Lissmann and Schwassmann 1965; Black-Cleworth 1970). More recent reports demonstrated the circadian nature of nocturnal variations in electric behavior, EOD-Br increases and social electric signals are more frequent during the night or subjective night (Engler and Zupanc 2001; Stoddard et al. 2007), provided social context is maintained. Due to EOD’s joint role as the carrier of sensory stimuli and communicative signals, changes in EOD rate show different states of awareness, novelty detection, social engagement and attention across different Gymnotiform species (Caputi et al. 2003; Silva et al. 2007; Jun et al. 2014, 2016; Perrone and Silva 2018; Gascue, V. et al. 2020). Behavioral recordings in G. omarorum have shown that the increase in EOD-Br is a natural trait, independent of exploratory behavior and persistent under natural constant darkness conditions (Migliaro et al. 2018).
Our results show that the electric behavior of B. gauderio, recorded in the natural habitat, has a daily rhythm of EOD-Br characterized by a steep increase in the afternoons, with a peak close to sunset, and a slow decay during the night towards sunrise. Individual acrophases are synchronized with the moment of maximal water temperature values, similarly to the behavior previously described for G. omarorum (Migliaro et al. 2018). Given the nocturnal habits of these fish, EOD-Br is expected to increase at night, as we observe in our results. Our data also shows another behavioral trait associated with the night, the increase in EOD-Br variability in both global and cycle to cycle measurements. This increase in rate variability reflects a wider range of inter-EOD interval duration (and hence of instantaneous frequency values), which is to be expected if animals are interacting and exploring their environment. It is interesting to consider whether this nocturnal increase in variability reflects an endogenous mechanism or is a consequence of the nocturnal increase in activity and awareness. EOD-Br variability has been reported to be very low in isolated resting gymnotiforms in laboratory conditions (Capurro et al. 1999b; Vitar 2019), contrasting with the natural daily modulation reported for G. omarorum (Gascue, V. et al. 2020) and for B. gauderio in the present work.
Information about the beginning and duration of the night is relayed to the vertebrate brain by the melatoninergic system. The circadian rhythm of endogenous melatonin production is synchronized with the night due to the inhibitory effect of the environmental light. In the present report we demonstrate that melatonin modulates electric behavior in both EOD-Br and EOD-BR variability. Moreover, electric behavior modulation is a consequence of the melatonin action on the discharge rate of the pacemaker nucleus and its variability. The similarity between the melatoninergic modulation of electric behavior and pacemaker activity suggests the central origin of this modulation. Melatonin decreases PM discharge rate and consequently EOD-Br, while increasing PN discharge rate variability with the concurrent increase in EOD-Br variability, showing an independent regulation of each trait.
Melatonin modulation of EOD-BR.
Our data shows that melatonin consistently decreases EOD-Br and that this influence is exerted at the level of the PN, through direct action on the in vitro preparation. This decrease in discharge rate seems at odds with the reported nocturnal increase in EOD-Br in both laboratory and natural conditions (Silva et al. 2007; Migliaro and Silva 2016). Moreover, melatonin is a key determinant of the nocturnal increase itself (Migliaro and Silva 2016). Melatonin actions on behavior and physiology are widely variable and context dependent. At the neuronal level melatonin has been reported to decrease excitability in different areas of the central nervous system as the cerebellum, suprachiasmatic nucleus or dorsal root ganglions through regulation of a delayed rectifier potassium current, modulation of GABAergic transmission, attenuation of the sodium current and also an interaction with passive membrane properties (Huan et al. 2001; Scott et al. 2010; Oliveira-Abreu et al. 2018). In this sense a decrease in the frequency of pacemaker activity is to be expected, as has been reported for other nuclei with oscillatory properties (Jiang et al., 1995; Mason and Brooks, 1988; Oliveira-Abreu et al., 2019; Scott et al., 2010; Shibata et al., 1989; Stehle et al., 1989). Moreover, this modulation has a time-of-the-day dependent outcome, due to circadian changes in the expression of melatonin receptors (Gaildrat et al. 1998; Ikegami et al. 2009; Feng et al. 2015). Mel1b receptor expression has been shown to be widespread in brain areas involved in vocal signaling in the midshipman fish (Feng and Bass 2016; Feng et al. 2019), a communication system which shares cellular, circuital and functional characteristics with the electrogenic system in fish. In this system melatonin receptors are expressed in bulbar nuclei as well as in upstream midbrain nuclei. This shows that melatonin regulation is a complex, multicomponent system with multiple pathways.
Electric behavior is the result of a number of influences converging in the PN that are in turn modulated by different factors with different temporal dynamics, among them are melatonin concentration and melatonin receptor expression. The buildup in the nocturnal melatonin concentration has species specific profiles across vertebrates (Falcón et al. 2010). In humans the physiological parameter indicating the timing of melatonin increase is called DLMO (dim light melatonin onset), and precedes the expression of nocturnal behavior in this diurnal species. The DLMO concept can be extended to other vertebrate species given the highly conserved features of the melatoninergic system. This holds true, even when environmental light is not a robust cyclic cue as happens with animals living in constant darkness or humans with retinohypothalamic tract damage (Cipolla-Neto and Amaral 2018). In this way the melatonin onset (MO) precedes the acrophase of a rhythmic behavior that is being modulated by melatonin itself. Hence, it is interesting to take a closer look at the natural nocturnal increase in EOD-BR, which has an acrophase at sunset that gradually declines towards sunrise. The melatonin onset might be working as a timing signal, affecting nuclei of the midbrain that are upstream from the PN, inducing the increase in EOD-BR. This increase will reach a maximum when the direct effect of melatonin on the PN starts counterbalancing the initial increase, generating the progressive decrease towards sunrise. This dual role as hormonal time giver and modulator of cellular excitability needs further confirmation, especially regarding melatonin onset in natural conditions.
Melatonin modulation of EOD-Br variability.
Gymnotiforms when isolated, unperturbed and resting are incredibly stable oscillators with respect to their electric behavior as well as to the spontaneous activity of the PN (Moortgat et al. 2000; Vitar 2019). This makes functional sense, as these animals require a robust system capable of withstanding external influences so as to not jeopardize their sensory capabilities. This stability however might be in conflict with the communication and cognitive capabilities of these animals, as using their EODs as a communication channel requires these animals to modulate their frequency in dynamic ways, usually within milliseconds after receiving a signal from a conspecific (Perrone et al. 2009). Exploring and navigating the world also demands rapid adjustments in EOD rate (Caputi et al. 2003; Jun et al. 2014).
In our behavioral experiments, melatonin produced an almost 10 time increase in the CV of EOD-Br. We can then assert melatonin’s role in the modulation of variability of electric behavior, mimicking the nocturnal increase in EOD-Br variability in the natural habitat. This increase in EOD-Br variability results from the increase in variability produced by melatonin in the central pacemaker, as shown in our results in comparison to saline treated preparations. An increase in variability allows for more flexible, rapid changes in the PN rate and hence in EOD-Br, as the ones that sustain social electric signals (Quintana et al. 2011, 2014; Comas et al. 2019).
This is the first report of the influence of melatonin on behavior in electric fish. In the present study we show a melatoninergic modulation of two traits of electric behavior, both of which must be under tight temporal regulation to allow for the correct behavioral display. This temporal association of processes through the action of the same modulator is common in systems where the temporal synchronization of multiple independent parts is crucial (Adkins-Regan 2013).