Reliability of behavioral test with fish: How neurotransmitters may exert neuromodulatory effects and alter the biological responses to neuroactive agents

Toxic agents such as pharmaceuticals and pesticides are continuously dispersed especially in the aquatic environment, as a result of human use. Their presence in the environment present serious concerns, since these compounds interfere with the normal function of the central nervous system (CNS), causing behavior alterations, whose consequences are difficult to predict. However, behavioral responses, even those that occur after exposure to neurotoxic agents, might be modulated by the release of neurotransmitters in the brain of exposed organisms, making even more difficult to ascertain the real consequences of pollution by neurotoxic or neuroactive agents. This study aimed to understand the potential of dopamine as neuromodulator in cases of acute exposure to a pesticide (the carbamate carbofuran) and to a therapeutic agent (the benzodiazepinic drug diazepam) in the freshwater fish Gambusia holbrooki . After acute exposure to both carbofuran and to diazepam it was possible to observe deleterious alterations in the motor function, reflected by significant reductions of both average speed and distance in exposed animals. These changes were later diminished and reverted by dopamine exposure. Despite the indications obtained from our experiments, more research is needed to clarify the consequences of these behavior alterations in a more integrative perspective, namely by adding behavioral endpoints of increased ecological relevance to the adopted experimental design.


Introduction
Journal Pre-proof presently.Bearing this need in mind, the critical overview by Legradi et al. (2018), comprehensively identified a series of critical bullet points concerning the future directions of the development of novel behavioral assessment methodologies to be used in ecotoxicological assessment of neuroactive compounds, as a way of predicting toxicity, identifying neurotoxic drugs, studying the contribution of mixtures of neuroactive chemicals, and allowing a real time measurement of neurotoxicity in the wild.However, the possibility that toxic effects may not only derive from exposure of living organisms to neurotoxicants alone, but rather be the conjugation of neurotoxicants and the complex modulatory effects caused by other neuroactive chemicals (including neurotransmitters), is still undiscussed.This is critical, since the main causes of behavior alterations include changes in the efficacy, function and regulation of the nervous system, which may be caused by the environmental exposure of biota to neurotoxicants, but also by other neuroactive compounds, of both exogenous and, most importantly to the present study, endogenous origin.
Environmental exposure to such agents is a common occurrence for many species (namely those of the aquatic environment), which leads to severe impairments of behavioral features in exposed biota (Kane et al., 2005).Neurotoxicity in aquatic organisms, for example, is commonly followed by changes in the nature and frequency of swimming movements (Little and Finger, 1990;Little and Brewer, 2001), which are of evident ecological importance, by conditioning common biological processes, such as feeding, predator avoidance, sexual activity, and camouflage.
neurotransmitter acetylcholine (Jash and Bhattacharya, 1982;Bhattacharya, 2001).This conditions leads to hyperexcitability of the nervous system, with fasciculation of nerve fibers; by acting this way, it compromises behavioral traits (Hernández-Moreno et al., 2011;Ibrahim and Harabawy, 2014;Saglio et al., 1996) Environmental exposure to this chemical has been implicated in malfunctions in the autonomic, somatic, and central nervous system (Aardema et al., 2008), causing behavioral effects, such as changes in the swimming activity, increased salivation, tremors and sedation, in teleost fish and in mammals (Barbieri, 2007a,b;Thörnqvist et al., 2019).
Another compound found in natural waters is diazepam (7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2-one).Diazepam is among the most used neuroactive drugs, all over the world, given its therapeutic properties, as a sedative and anxiolytic (Fallis, 2013).Diazepam belongs to the therapeutic class of the benzodiazepines, and is used as a muscle-relaxant, anti-convulsive, anxiolytic, sedative, and antiepileptic (Dhir and Rogawski, 2018;Lorenzi et al., 2006;Oggier et al., 2010).Diazepam acts pharmacologically by increasing the affinity of gamma-aminobutyric acid (GABA) receptors for their physiological ligand.This effects results in the enhancement of the inhibitory action of GABA molecules, by binding with increased affinity to their receptors, and thereby increasing the inwards flow of chloride ions, and reducing the excitability of resting neurons (Rudolph and Knoflach, 2011).This effect results in a decrease in the excitability of affected neurons, reducing anxiety, acting as a muscle relaxant and as a sleep inducer (Osswald and Moura, 2006).In fish, drugs of the benzodiazepinic class cause loss of control of normal behavioral functions, such as Journal Pre-proof J o u r n a l P r e -p r o o f 6 swimming (Montanha and Pimpão, 2012).The presence of this pharmaceutic drug has been already reported in effluents of German sewage treatment plants, in concentrations up to 0.04 µg L -1 , and in the Po River, Italy, in concentrations up to 2.13ng L -1 (Calamari et al., 2003;Ternes, 1998).
The previously mentioned compounds, alone, may alter behavior; however, their effects may be conditioned or modulated by the co-occurrence of neurotransmitters, whose levels may vary according to the biological fluctuations of exposed organisms.
For example, in stressful situations neurotransmitters such as gamma-aminobutyric acid, dopamine, norepinephrine, serotonin, melatonin, and glutamate, actively participate in counteracting the effects caused by the stress evoked episodes (Kumar et al., 2013).Despite the undisputable importance of the above mentioned neurotransmitters in maintaining the homeostatic balance, dopamine can mask or otherwise alter behavioral traits in unexpected ways.For example, (Ungerstedt, 1971), showed that dopamine-deprived rats were unable to show motivation for feeding behavior, compromising their survival.So, the dopaminergic system has a role in reward, reinforcement, but also in stress responses (Øverli et al, 2001;Cabib and Puglisi-Allegra, 2012;Koob and Volkow, 2016;).Normally large doses of dopamine are released in the brain when animals present a higher level of stress (Cabib and Puglisi-Allegra, 2012).Data from the literature show that dopamine is released in stress-induced situations to promote responses of avoidance and removal from the stressful situation (Cabib and Puglisi-Allegra, 2012).Therefore the dopaminergic system is extremely important for adaptive and behavioral responses when organisms are facing new environmental stimuli, being highly evolutionarily conserved (Tay et al., 2011).There are some evidences establishing a connection between the Journal Pre-proof J o u r n a l P r e -p r o o f mesolimbic dopamine system with a social network, altering aggression, reproduction, spatial learning, emotional learning and parental care patterns (O'Connell and Hofmann, 2011).Although highly conserved, the dopaminergic system, may be altered by other neurotransmitters and by neurotoxins.For example, there are cholinergic projections on one of the major sources of dopamine (ventral tegmental area) also leading to fluctuations in dopamine mediating uncertaintyseeking behaviors (Avery and Krichmar, 2017;Belkaid and Krichmar, 2020;Scatton et al., 1980).The dopaminergic system can also be alter by the neurotoxicity of several insecticides (e.g.fipronila GABAergic insecticide) (Bharatiya et al., 2020).
Previous studies showed that a decrease in dopaminergic neurons, elicited by the compound above, is associated with deficits in spontaneous motor activity, in motor coordination, in memory recognition and social interaction ( Jackson et al., 1983;Lee et al., 1996;Deumens et a., 2002;Ferro et al., 2005;Bové and Perier, 2012;) Therefore, dopamine (and the modulation of the dopaminergic neurotransmission pathway) can have multiple and unexpected effects on behavior, e.g. can stimulate aggression, increase impulsivity and activate other responses.These effects seem not to be restricted to endogenous dopamine, and exogenous sources of this neurotransmitter seem also to cause similar effects.Screbina, in 2012, showed that, individuals of zebrafish exposed to a dopamine (D1-R) antagonist (SCH23390), decreased their shoaling behavior.It is thus possible to suggest that dopamine, exogenous or endogenous, may alter the toxic effects on behavior caused by environmental exposure to potential neuroactive compounds, such as pesticides acting on the central nervous system, and drugs, such as tranquilizers, despite the differences in their chemical nature and potentially compromised neurological pathways.
Journal Pre-proof J o u r n a l P r e -p r o o f 8 Fish, among aquatic organisms, are ideal for behavioral assessments since are in direct contact with water putatively contaminated with the chemicals under study (Kane et al., 2005).Considering their wide number and natural abundance, being distributed along a series of aquatic ecosystems, fish are of geographical and ecological relevance, being also easy to maintain in laboratory conditions (Kane et al., 2005;Little et al., 1993).Poeciliidae such as Gambusia holbrooki, have a large potential as a test organism of environmental contamination (Osten et al,.2005;Freitas and Siqueira-Souza, 2009).In addition, fish behavior has been thoroughly used as an effect criteria in ecotoxicological studies.This use is justified considering that fish, in general, tend to exhibit a typical behavior.Normally, fish have a natural tendency to dive to the bottom, when introduced into a new environment, and then, tend to increase their swimming activity, vertically, over time (Kalueff et al., 2016;Thörnqvist et al., 2019).But when exposed to neurotoxicants, such as insecticides, some fish species have demonstrated avoidance behavior (Kynard, 1974).Also, when subjected to highly stressful situations, or during drug-evoked anxiety episodes, fish tend to reduce their activity at the surface, while increasing their movement in bottom areas.
In this study we aimed to understand the effects of exogenous dopamine on behavior (namely, swimming behavior) of individuals of the freshwater poecilide species Gambusia holbrooki, after being exposed to the neurotoxic chemicals carbofuran and diazepam.

Test organisms
Individuals of the test species, G. holbrooki, were manually captured in a local freshwater pond, with hand nets.Fish were immediately sorted after capture, by sex and maturation stage; only adult males were kept, and females were discarded (potential hormonal fluctuations involved in the reproductive cycle of females could biased the obtained data; Ericsson et al., 2019).Sexually immature fish and mature females were them released in their natural environment.After capture, fish were transported to the laboratory, and housed in aquaria with dechlorinated tap water, weekly exchanged, with mechanical filtration and continuous aeration.The organisms were kept at 21 ± 1ª C and with a 16:8 light/dark photoperiod; pH and ammonia were also monitored.Fish were fed once a day with TetraMin® flakes, ad libitum.The organisms were kept for 15 days for depuration/acclimation/quarantine under controlled conditions, before trials.

Experimental design -exposure
The experimental design involved exposing 10 fish, each one in 250 ml flasks with continuous aeration, per tested condition.In our control (C) the test organisms were Journal Pre-proof J o u r n a l P r e -p r o o f 10 exposed to dechlorinated tap water (250ml per individual).Group CBF and DZP, also with 10 individuals per trial, were separately exposed to carbofuran and to diazepam, respectively.Group DOP was exposed only to dopamine.Finally group CBF-DOP and DZP-DOP (individual exposure of 10 individuals per group) tested the behavior alterations when fish were initially exposed to carbofuran and them to dopamine (CBF-DOP), and when fish were primarily exposed to diazepam and them to dopamine (DZP-DOP).The organisms were kept with continuous aeration, at 21± 1ª C with a 16:8 light/dark photoperiod; all the media were changed after 48h.Ammonia and pH were also monitored.
The compounds carbofuran, diazepam, and dopamine were diluted in dechlorinated tap water, to achieve levels of 165mg/L, 1.2 mg/L, and 1 mg/L, respectively.These concentrations correspond to the calculated LC 10 values for both drugs, published in previous works (Nunes et al., 2005;Osten et al., 2005;Scerbina et al., 2012).Dopamine can be administered in various ways.In this work, we diluted dopamine in the exposure media, since previous studies (Mok andMunro, 1998 andScrebina et al., 2012) demonstrated that this compound could produce effects by being administered by this route, without the need to injure or stress the organisms.After 96h of exposure, all organisms from the groups C, CBF, DZP, and DOP, were removed, and were released in a 1.5L tank, and their behavior was analyzed by monitoring and recording their movements for a period of 5 min.For the tests CBF-DOP and DZP-DOP, each organism was wept, for 96h, in media contaminated by the compounds carbofuran and diazepam; after this period of exposure, fish were removed from exposure apparatuses, and were exposed to dopamine for an additional period of 30 Journal Pre-proof min (Screbina et al., 2012).Finally, their behavior was analyzed and monitored following the same procedures described above.

Behavioral assessment
The behavioral assessment was undertaken by adapting glass aquaria to this purpose.
Each tank was divided in 3 zones, to measure the time spent by the fish in the different zones.The 3 zones corresponded to the a) 'bottom' zone (the first 3,5cm of the aquarium, measured from the 'bottom'); b) 'upper' zone (the following 3,5cm of the aquarium, measured from the end of the 'bottom' zone); and c) the 'surface' zone (the last cm of the 'upper' zone) (fig.1).We decide to establish this 'surface' zone to evaluate minor and finer movements, since this species (a poecilide) is known to live and prey at the top (surface) of the water column (FishBase, 2004;IGGS, 2006;Adey and Loveland, 2007).To avoid disturbances caused by the operator, all sides of the assessment tanks were covered with white opaque sticker paper.Between the behavioral assessments of different individuals, the aquarium was cleaned with tap water and ethanol 90%.Trials involved recording the behavior of each individual fish at 30 frames per second with a camera (Xiaomi Redmi note 4).The previously recorded swimming behavior was analyzed using a free image software, Toxtrac (which provides x and y metrics of every organism path) (Rodriguez et al., 2017;Rodriguez et al., 2018) and a software developed by our work team (for more information, see the footnote below) 1 , which allowed calculating values of mean we evaluated the effects of neuromodulation with dopamine after the exposure to carbofuran and diazepam, separately, by using a t-test (Fig. 3).

Discussion
Behavior alterations are triggered in animals exposed to challenging conditions, such as the environmental presence of contaminants.Despite being an individual response, the final stage of a response to such stressful conditions is characterized by an abnormal behavior, with potential deleterious impact in the community (Kane et al., Journal Pre-proof J o u r n a l P r e -p r o o f 14 2005).Fish display complex responses when facing stressful episodes, and that dopamine is instrumental for the physiological regulation of stress-related responses.
In teleosts, dopamine can enforce varying effects, conditioning inhibitory or excitatory responses.It is however to considerer that the neurological effects of dopamine are not limited to the dopaminergic system; in fact, they go well beyond the merely activation of the dopaminergic circuits.Their effects (even after exogenous administration) for instance, can also exert effects on GABAergic pathways, as demonstrated by Lou et al. (2016).This assumption evidences that despite being primary acting on distinct and already studied biochemical pathways, their influence may occur also on other routes, with never before studied consequences, namely at the behavioral level.In addition, chronic exposures to dopamine may exert an inhibitory effect on the release of gonadotropins, affecting the sexual development and maturation (Rezende, 2013).Nevertheless, and most importantly for the present study, the dopaminergic system in fish also plays a fundamental role in brain function, such as motor function (Mok and Munro, 1998).Some studies have shown that in fish exogenous dopamine leads to an increase in locomotor activity (Mok and Munro, 1998) and alterations in shoaling behavior (Scerbina et al., 2012).
The swimming behavior is frequently used in toxicological research because changes in the locomotor activity often reflect an effect on the nervous system (Brewer et al., 2001), i.e., neurotoxicity becomes evident when movement is compromised.
Neurotoxicity can be caused by alterations of many pathways.In this work we highlighted the effects elicited by an anticholinesterase agent (carbofuran) and a tranquilizer agent (diazepam).These two compounds may have effects on nerve and muscle cells, despite the putative differences in their mechanisms of toxic action.On Journal Pre-proof J o u r n a l P r e -p r o o f 15 one hand, the anticholinesterase agent (carbofuran) leads to an inhibition of cholinesterases (namely of acetylcholinesterase), overstimulating the muscular cells by favoring the accumulation of the neurotransmitter acetylcholine at the synaptic cleft (Bhattacharya, 2001).On the other hand, the tranquilizer agent (diazepam) acts by favoring the activity of the neurotransmitter GABA at its receptors, reducing the excitability of resting neurons and consequently leading to a hypo excitability of the central nervous system (CNS) (Overturf et al., 2016).Despite not being apparently connected to effects on the dopaminergic route, exposure of fish to both chemicals resulted in behavioral effects, which were somehow changed when animals were also exposed to dopamine.
From our results, the occurrence of a decrease in the parameters average speed and distanced moved was made clear, in fish exposed both to carbofuran and to diazepam, compared to animals from the control treatment.This decrease was somewhat expected considering the already described effects based on the mechanisms of toxic action of both chemicals.Despite the differences in the involved pathways, both chemicals were able to compromise neuromuscular functioning, thus leading to reduction in movement of exposed organisms, not only in fish but also in mammals.
These effects were already reported in other organisms besides fish (Oswald, 2001;Barbieri, 2007 a,b;Papich and Papich, 2016), such as cats (Cotler et al., 1984), dogs (Musulin et al., 2011), and rodents (Dhir and Rogawski, 2018).Whit this in mind, it is possible to suggest alterations in feeding, predatory, and sexual behavior leading to population effects, which bear ecological relevance.
In the specific case of carbofuran, behavioral impairment may occur as a consequence of its involvement in the deleterious effect in peripheral processes of muscular Journal Pre-proof J o u r n a l P r e -p r o o f 16 communication, but also as a consequence of its central effects, which can result in loss of muscular control.It has already been demonstrated that the central nervous system may be damaged by the inhibition of acetylcholinesterase, thus affecting locomotor activity (Smith, 1984).Brewer (2001), anticipated a biphasic response in the swimming behavior of fish, when animals were exposed to organophosphate insecticides, namely malathion.Initially, exposure to these chemicals may cause an excitatory response, with an increase in distance and speed, shortly followed by a decreasing phase, as a consequence of the inhibition of cholinesterase activity, during which the behavioral activity was depressed.Despite the absence of clear indication on the occurrence of this biphasic response, our results partly corroborate the previous study, also demonstrating a decrease in average velocity and distance moved when the organisms were exposed to the pesticide carbofuran.
The here-observed effects of diazepam are likely to be related to its pharmacological activity, which are often involved in behavioral modifications.This therapeutic agent is able to suppress anticonvulsive contractions in mammals (Musulin et al., 2011), and has been shown to have sedative and hypolocomotor effects, preventing a decrease in social interactions in amphetamine withdrawal situations (Rincón-Cortés et al., 2018).
The sedative effects of diazepam, mediated by enhancement of inhibitory GABAergic action, have shown to reduce anxiety-like behavior (Bencan et al., 2009).For example, zebrafish individuals when exposed to a new environment, exhibit an initial anxiety-like diving response.But when pre-treated with diazepam, this response was attenuated (Bencan et al., 2009).The deleterious effect of this compound, has also been reported in other species, like convict cichlid (Amatitlania nigrofasciata), resulting in a reduction of stress-related behavior when exposed to new environment Journal Pre-proof J o u r n a l P r e -p r o o f (Hope et al., 2019).In addition, Nunes et al (2008), reported that poecilids of the same species used in our work, G. holbrooki, when exposed to diazepam, showed lethargic behavior potentially associated to alterations of the GABAergic neurotransmission.
The alterations previously mentioned, may bring adverse consequences to the species involved, but also to the population due to changes in locomotor activity, which may result in alterations of feeding, sexual and social behavior (Snigirov and Sylantyev, 2018).
Despite the absence of effects reported in animals exposed only to dopamine, the association of both toxicants and dopamine, yielded distinct behavioral profiles.It thus seems that exogenous dopamine alone is not capable of significantly altering the here observed behavioral endpoints.However, previous data from the literature suggested this possibility; according to Screbina et al (2012), exogenous administration of this neurotransmitter has been also involved in behavioral alterations, due to the hiperpolarization of nerve cells caused by this cathecolamine.
However, this hypothesis was not confirmed by our data, since animals exposed to dopamine alone had not their behavior changed when compared to control fish.
Nevertheless, the association of carbofuran/diazepam with dopamine was capable of significantly altering the behavior of fish.In fact, when testing the neuromodulatory effects of dopamine, we observed an increment in the velocity and distance traveled, when compared to the organisms exposed only to carbofuran or diazepam.We previously showed that exposures to carbofuran and diazepam had a deleterious effect on velocity and distance traveled, but these effects were reverted after exposure to dopamine.Dopamine thus seemed to have enhanced the locomotor activity of exposed fish.This effect may be due to the relation between dopamine and movement control.Exposure to a dopamine agonist has shown enhanced locomotor activity in individuals of two fish species, namely Oreochromis niloticus and O. mossambicus (Mok and Munro, 1998).Therefore, an increment in the speed and distance traveled was expected, and this effect indeed occurred in our test organisms.
Our results did not demonstrate any significant differences in the behavior of spatial positioning in the water which was assessed by measuring the time spent in the 'bottom' zone, in the 'upper' zone and in the 'surface' zone, and for all the exposed groups.Consequently, we may assume that the bidimensional preference of fish was not affected by any of the treatments.This is paradoxical, since in other studies with zebrafish (Danio rerio), the tested organisms showed a different behavior towards their positioning in the water column (e.g.Kalueff et al, 2016).When exposed to anxiolytic drugs, fish had an increased 'surface' swimming, but when exposed to high stress-or drug-evoked anxiety, fish tended to increase 'bottom' swimming.Fish of the same species here used, G. holbrooki, were already demonstrated to be responsive when in the presence of diazepam, with significant changes in their behavior; the study by Nunes et al (2008) reported that fish tended to swim at the surface of the exposure apparatuses.This increased 'top' swimming was however caused by an expansion of the swim bladder (Nunes et al., 2008), an effect that might be related to the GABAergic effect of benzodiazepines.The swim bladder of some fish species is an organ controlled by parasympathetic innervation (inflation) and sympathetic innervation (deflation) (Smith and Croll, 2011).However, there are some evidences that dopaminergic pathways and GABAergic fibers may also be involved in the control of this organ (Nunes et al., 2008;Rezende, 2013;Smith and Croll, 2011).Differences in our results may be due to species-specific patterns of Journal Pre-proof J o u r n a l P r e -p r o o f 19 toxicity and differences in the tested dosages, which were in this case much lower than those described in the literature as being causative of such modifications in the swim bladder inflation (Kane et al., 2005;Nunes et al., 2008).

Conclusions
Due to its therapeutic properties, diazepam is among the most worldwide prescribed drugs, and is likely to reach the aquatic environment, interacting with exposed fauna.
In addition, other neurotoxic agents, namely of agricultural use, such as carbofuran, are employed and dispersed in massive amounts, endangering non target species.
From our results, we can conclude that, even in small amounts, carbofuran and diazepam were responsible for a deleterious effect on Gambusia holbrooki nervous system, affecting the motor function of exposed fish.Also, we could infer significant reversion of such changes in motor function, with the increment of dopamine after acute exposure to both carbofuran and diazepam.It is possible to suggest that the neuromodulatory effects of specific neurotransmitters may however act as confounding factors in the interpretation of these behavioral assessments.

Figure 2 -
Figure 2 -Mean velocity (mm/s), average distance moved (mm), time spent in the

Figure 3 -
Figure 3 -Mean velocity (mm/s), average distance moved (mm), time spent in the