n ± S.D. of six larvae.
Risperidone treatment at 4 dpf only transiently decreased SSA, with full recovery after 7 dpf.
SSA was significantly below that of controls (Figure 2(b)) in the first days after treatments (6 dpf, P < 0.05). After 7 dpf, SSA had recovered to control levels, these levels were maintained throughout the remaining experimental period (Figure 2(b)).
The same occurred in zebrafish treated with 5 μM risperidone at 6 dpf and monitored at 8 dpf (8 dpf, P < 0.05).
A 24-h exposure to fluoxetine at 4 dpf resulted in a significant decrease in SSA as compared to controls (6 dpf, P < 0.01). SSA recovered fully after 7 dpf, and increased significantly at 9 dpf (9 dpf, P < 0.01) (Figure 2 (b)); however, the movements were erratic and abnormal. It is worth mentioning that when fish were exposed to fluoxetine at 4 dpf and then to risperidone at 6 dpf, there was no significant change in SSA with respect to control (Figure 2(b)).
3.3. Tissue Sections
3.3.1. Morphological Changes
Both treated and control animals were fixed at 10 dpf, cut in serial sections and stained, as mentioned above. A 24-h exposure to fluoxetine at 4 dpf resulted in an observable decrease in the number of nucleated areas of the dorsalis telencephali area. The same occurred in the dorsal area of the optic tectum (Figure 3(b)) compared with the control (Figure 3(a)).
There was also a larger area in the postoptic commissure and the raphe population zone and a cellular disorganization in the latter. On the other hand, the fissure that separates the midbrain at forebrain increased in size (Figure 3(b)). All these changes were reflected as an increase in abnormal and erratic swimming in the behaveioral test. Undoubtedly, the administration of this drug to animals at 4 dpf caused dramatic changes that persisted overtime.
In the treatment with risperidone at 4 and 6 dpf (Figures 3(c) and (d)), an increase in the nucleated zone of
Table 1. Post-treatment heart rates in zebrafish larvae after a 24 h exposure to 5 μM Fluoxetine or risperidone. A mean number of heart beats per minute (bpm) (±S.D.) in control and 24 h fluoxetine or risperidone treated zebrafish larvae. There was no significant difference between control and fluoxetine or risperidone exposed larvae at either 8 or 9 dpf (P > 0.05, n = 6).
Figure 2. (a) Schematic representation of a 6-well multiplate with a dividing line for monitoring the spontaneous swimming activity (SSA); (b) Spontaneous swimming activity (SSA) measurements. All SSA measurements began at 6 dpf and were performed three times; each data point represents the mean ± S.D. of six larvae.
the postoptic commissure and a cellular disorganization in the raphe populations zone were observed. However, these changes would not be sufficient to affect the SSA.
Zebrafish exposed to fluoxetine at 4 dpf and then to risperidone at 6 dpf showed no significant changes with respect to control (Figure 3(e)).
For the histological analysis, crop images were obtained to include the reference space area and optical angle for brain tissue. In this work, we studied two proteins to determine dopaminergic neurons, labeled with TH, and motoneurons, labeled with CalR.
When the larvae were exposed to free risperidone, an increase in the levels of TH + dopaminergic neurons was observed in the dorsal region of the optic tectum of the brain (Figures 4(c) and (d)) compared with the control (Figure 4(a)). On the other hand, increases in the levels of TH + cells in the midbrain region were also observed. However, no significant changes in brain tissue (Figures 4(b) and (e)) were observed with fluoxetine either alone or combined with risperidone.
In the spinal cord, we found a decrease in the levels of CalR-positive motoneurons in all treatments with risperidone alone (Figures 5(c) and (d)) compared with the control (Figure 5(a)). In the case of fluoxetine, an increased width in the nerve fiber formed by the axons of motoneurons (Figure 5(b)) was observed. In the case of fish treated with the combination of fluoxetine and risperidone, there was an increased width in the nerve fiber formed by the axons of motoneurons compared with those treated with risperidone (Figure 5(e)) but less than that found in those under fluoxetine alone.
Figure 3. Images of histological sections of brain tissue stained with hematoxylin-eosin. (a) Control; (b) Fluoxetine at 4 dpf; (c) Risperidone at 6 dpf; (d) Risperidone at 4 dpf; and (e) Fluoxetine at 4 dpf and risperidone at 6 dpf. Larvae were analyzed three times (n = 3) at 10 dpf. In Figure 3(b), the arrows indicate an observable decrease in the number of nucleated areas of the dorsalis telencephali area and the optic tectum; also a larger area in the postoptic commissure and the raphe population zone and a cellular disorganization in the latter. On the other hand, the fissure that separates the midbrain at forebrain increased in size.
Figure 4. Immunohistochemistry images of brain tissue. Tyrosine hydroxylase, labeled with Cy2 (green) and Calretinin labeled with Cy3 (red). (a) Control; (b) Fluoxetine at 4 dpf; (c) Risperidone at 6 dpf; (d) Risperidone at 4 dpf; and (e) Fluoxetine at 4 dpf and risperidone 6 dpf. Larvae were analyzed three times (n = 3) at 10 dpf. The arrows in Figures 4(c) and (d) indicate an increase in the levels of TH + dopaminergic neurons in the dorsal region of the optic tectum of the brain and an increase in the levels of TH + cells in the midbrain region.
Figure 5. Immunohistochemistry images of spinal cord tissue. Tyrosine hydroxylase, labeled with Cy2 (green) and Calretinin labeled with Cy3 (red). (a) Control; (b) Fluoxetine at 4 dpf; (c) Risperidone at 6 dpf; (d) Risperidone at 4 dpf; and (e) Fluoxetine at 4 dpf and risperidone at 6 dpf. Larvae were analyzed three times (n = 3) at 10 dpf. In Figures 5(c) and (d), the arrows indicate a decrease in the levels of CalR-positive motoneurons. In Figures 5(b) and (e) the arrows indicate a change in the width of the nerve fiber formed by the axons of motoneurons.
In this work, the effects of risperidone and fluoxetine on the locomotor activity, heart rate, and brain development of zebrafish larvae were studied. These parameters provide an idea of the effects caused by serotonin in early development stages and provide knowledge on the area of neuropharmacology.
The advantage of studying motoneuron diseases in zebrafish  is the rapid development of their spinal cord, which allows the analysis of motoneuron branching patterns as early as 24 hpf. In addition, responses to touching and swimming can be monitored after hatching around 48 hpf . It is now recognized that zebrafish show great similarity to mammals and are an extremely useful model for screening compounds at several stages of the drug discovery process [27,28].
The effect of fluoxetine and risperidone exposure was evaluated by examining the heart rate. The results demonstrated that 5 μM fluoxetine and/or risperidone-treated larvae exhibit a normal heart rate at 8 and 9 dpf compared to controls. When larvae were exposed to 5 μM fluoxetine and/or risperidone at 4 and 6 dpf and for 24 h, SSA was monitored daily from 6 to 9 dpf. SSA was significantly lower than controls on the first days after treatments. After 7 dpf, SSA levels recovered up to those of control, and were maintained throughout the remaining experimental period under risperidone treatment. In larvae exposed to 5 μM fluoxetine alone, SSA was significantly increased at 9 dpf as compared to controls. However, erratic and abnormal movements were observed with this increase, suggesting a long-term toxic effect of fluoxetine. The unusual body position exhibited in swimming during the behavioral test after exposure to fluoxetine was also observed. While control animals maintained a parallel position with respect to the water surface when swimming, the animals treated with fluoxetine were unable to maintain such a position, indicating lack of postural balance, as described in previous studies . In addition, we found that fluoxetine-treated zebrafish exhibited erratic swimming patterns, manifested by bouts of vertical swimming or sideway swimming, suggesting a coordination problem. This extended effect suggests that fluoxetine effect is not pharmacological, but rather developmental. However, when fish were treated with fluoxetine and risperidone, SSA showed no significant differences with control, and no erratic swimming was observed. This result suggests a reversible process, given that fluoxetine increases the levels of serotonin and risperidone levels out this effect. So, it is important to note that changes in SSA are not pharmacological because for extended periods of time, the effects are reversed when the concentrations of the neurotransmitter are decreased.
Representatives from all major fish classes possess serotonergic neurons in the raphe nuclei, giving rise to ascending and descending pathways [18,30-47]. Due to their apparent resemblance with the mammalian raphe serotonergic neurons, these are the best studied populations within fish. Generally, the assumption for teleosts has been that the two raphe nuclei (superior and inferior raphe) innervate most brain areas in a manner similar to that described for mammals and, accordingly, that they may play equivalent functional roles. Indeed, 5-HT immunoreactive fibers have been detected throughout the fish CNS [30,33,37,39,41,42,48-51].
The raphe serotonergic innervation of the zebrafish dorsal telencephalon, in particular of the lateral zone, is very dense. Further, Lillesaar et al.  observed few serotonergic fibers in the medial zone of the dorsal telencephalon originating from the raphe fibers. Interestingly, developmental studies in zebrafish as well as axonal tracing and lesion experiments combined with behavioral assays performed in goldfish suggest that these areas are functionally equivalent to the mammalian hippocampus and amygdala, respectively [52-54]. In mammals, the hippocampus is involved in spatial, contextual, or relational memory and it is known to be supplied by a rich serotonergic innervation mainly from the median raphe populations [55,56].
Several antipsychotic drugs cause a neurotoxic mechanism resulting from an increased or decreased concentration of serotonin both in the synaptic and extracellular spaces. In this sense, drug exposure at 4 or 5 dpf coincides with the initial appearance of inferior raphe axons distributed throughout the entire length of the spinal cord in zebrafish . Growth cones of these axons at 4 dpf have been observed adjacent to reticulospinal neurons in the hindbrain and secondary motoneurons in the spinal cord. The temporal correlation between the growth of inferior raphe axons and growth cones throughout the spinal cord and the earliest morphological effects of antipsychotics drugs suggest that raphe axons are affected by the exposure to these drugs. The mechanism of excess or default of serotonin toxicity has been elusive. Considering the temporal correlation between the development of the inferior raphe pathway and risperidone exposure, it was surprising to find no change in SSA. The transient depression of SSA in larvae exposed to risperidone at 4 and 6 dpf may be due to the long half-life of the drug . When the larvae were exposed to fluoxetine alone, first a decrease in swimming and then an increase with erratic movements were noted. However, in zebrafish exposed to fluoxetine at 4 dpf and then to risperidone at 6 dpf, there was no significant change with respect to control, indicating a possible reversal of the effects caused by exposure to fluoxetine observed previously.
In immunohistochemical assays, we used two proteins to determine dopaminergic neurons, labeled with tyrosine hydroxylase (TH) and motoneurons, labeled with calretinin (CalR). TH is the first enzyme in the catecholamine synthesis pathway . TH has also been described to be involved in the stabilization of dopamine synapses
. When the larvae were exposed to free risperidone, several changes in the brain were observed. This could be because the drug is a strong blocker of dopamine receptors, which would affect dopaminergic neurons. However, despite the changes observed in the distribution pattern of dopaminergic neurons, these would not affect the SSA. On the other hand, when administered fluoxetine alone or fluoxetine and risperidone, there were no changes with respect to the control. These results indicate that risperidone alone increased the levels of TH enzymes of the dopaminergic cells when administered as a single drug.
CalR was found in several neuronal populations of the central and peripheral nervous system, primarily in motoneurons and other sensory pathways . CalR is a cytosolic protein of 29-kD, belonging to the family of calcium-binding proteins “EF hand” , these expressed during early development of the CNS of vertebrates , although CalR needs some degree of cell differentiation and tissue to be expressed . Calcium-binding proteins buffer intracellular calcium, thus contributing to the properties of neuronal membranes and their electrical activity. In the spinal cord, we found a decrease in the number of CalR-positive motoneurons in all treatments with free risperidone. These changes could be due to the effect of risperidone on the core neural migration raphe (located in the hypothalamus) to the spinal cord. This migration begins to be observed between 2 and 3 dpf and extends into the caudal spinal cord after 4 days . In the case of fluoxetine, an increase in the width of nerve fiber formed by the axons of motoneurons was observed. This could cause a significant increase in SSA at 9 dpf with erratic and abnormal movements, given the potential neurotoxicity of the drug. In this respect, antipsychotic drugs could alter the extracellular levels of neurotransmitters and thereby modify the development of the CNS [7,14,28,64]. These changes suggest that the neuroanatomy is affected by fluoxetine and risperidone exposure, since these changes are not sufficient to modify the SSA in the case of risperidone.
According to Seibt et al. , antipsychotic drugs show high affinity for biomembranes due to their amphiphilic property. This implies that antipsychotic drugs can also interact with membrane lipid organization. The intercalation of antipsychotic drug molecules into the plasma membrane can modify the membrane lipid dynamics, inducing a subsequent modification of the receptor response . Drug interaction elicits short and long range inﬂuence on the bilayer structure, conesquently, modulating processes that range from membrane-bound enzyme activity and receptor binding to membrane permeability and transport . The intercalation of antipsychotic drug molecules into the plasma membrane may thus modulate the efficacy and tolerability profile of compounds able to exert their therapeutic effect through their binding with synapse receptors. This suggests that the pharmacological activity of antipsychotic medications may result from a combination of drug-receptor and drug-membrane interactions . Giacomini et al.  have suggested that the pharmacological actions of these antipsychotic drugs may be well retained in vertebrates.
Studies in zebrafish will provide an important insight into the side effects of these drugs as well as into the brain control of locomotor activity.
Testing several drug-induced changes in behavioral and serotonin levels is one of the experimental approaches for screening a new therapeutically relevant compounds, and merits further research in this animal model.
This research was supported by a grant from Universidad Nacional de Quilmes. Silvia del Valle Alonso is a member of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and IMBICE, CICPBA (Provincia de Buenos Aires, Argentina). M. J. Prieto acknowledges fellows from CONICET, Argentina.
We would also like to thank Instituto de Neurociencias de Castilla y León, Facultad de Medicina, Universidad de Salamanca, España, as well as Universidad Nacional de Quilmes and Ministerio Nacional de Ciencia, Tecnología e Innovación Productiva (MINCYT) grants.
Hpf: Hours Post Fertilization;
Dpf: Days Post Fertilization;
SSA: Spontaneous Swim Activity;
TH: Tyrosine Hydroxylase;
CNS: Central Nervous System;
ASD: Autism Spectrum Disorders;
SSRI: Selective Serotonin Reuptake Inhibitor;
LOEC: Lowest Observable Effective Concentration;
bpm: Beats Per Minute;
PB: Phosphate Buffer 0.1 M;
PFA: 4% v/v Paraformaldehyde;
RT: Room Temperature;
MLF: Medial Longitudinal Fascicle.