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Accepted Manuscript
Title: Ivermectin reduces motor coordination, serum
testosterone, and central neurotransmitter levels but does not
affect sexual motivation in male rats
Authors: N. Moreira, T.M. Sandini, T.M. Reis-Silva, P.
Navas-Suáresz, A.V.V. Auada, I. Lebrun, J.C. Flório, M.M.
Bernardi, H.S. Spinosa
PII:
DOI:
Reference:
S0890-6238(16)30435-X
https://doi.org/10.1016/j.reprotox.2017.10.002
RTX 7593
To appear in:
Reproductive Toxicology
Received date:
Revised date:
Accepted date:
18-11-2016
3-10-2017
17-10-2017
Please cite this article as: Moreira N, Sandini TM, Reis-Silva TM, Navas-Suáresz
P, Auada AVV, Lebrun I, Flório JC, Bernardi MM, Spinosa H.S.Ivermectin
reduces motor coordination, serum testosterone, and central neurotransmitter levels
but does not affect sexual motivation in male rats.Reproductive Toxicology
https://doi.org/10.1016/j.reprotox.2017.10.002
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1
Ivermectin reduces motor coordination, serum testosterone, and central
neurotransmitter levels but does not affect sexual motivation in male rats
Moreira, N.a; Sandini, T. M.b; Reis-Silva, T. M.c; Navas-Suáresz, P. a; Auada, A. V. V.e;
Lebrun, I.e; Flório, J. C.d; Bernardi, M. M.f; Spinosa, H. S.d
a
Graduate Program of Experimental and Comparative Pathology, Department of Pathology,
School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr.
Orlando Marques de Paiva, 87, São Paulo, SP, 05508-270, Brazil.
b
Graduate Program of Toxicology and Toxicological Analysis, School of Pharmaceutical
Science, University of São Paulo, São Paulo, Brazil. Av. Prof. Dr. Lineu Prestes, 580, São
Paulo, SP, 05508-000, Brazil.
c
Graduate Program of Neuroscience and Behavior, Department of Neuroscience, Institute of
Psychology, University of São Paulo, Av. Prof. Dr. Melo de Morais, 1721, São Paulo, SP,
05508-030, Brazil.
d
Department of Pathology, School of Veterinary Medicine and Animal Science, University
of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, São Paulo, SP, 05508-270, Brazil.
e
Laboratory of Biochemistry and Biophysics, Butantan Institute, Av. Vital Brasil, 1500, São
Paulo, SP, 05503-900, Brazil.
f
Graduate Program of Environmental and Experimental Pathology and Graduate Program of
2
Dentistry, Paulista University, R. Dr. Bacelar, 1212, São Paulo, SP, 04026-002, Brazil.
Conflict of Interest Statement: All authors declare that there are no conflicts of interest.
Source of funding: CAPES, FAPESP (Process nº 2015/03131-4), CNPq (Process nº
305500/2013-9).
Highlights
 Ivermectin impaired sexual behavior and motor coordination through GABAergic
activation.
 Ivermectin did not affect sexual motivation or penile erection.
 Ivermectin reduced the levels of serum testosterone, GABA, and striatal dopamine.
Abstract
Ivermectin (IVM) is a macrocyclic lactone used for the treatment of parasitic infections and
widely used in veterinary medicine as endectocide. In mammals, evidence indicates that IVM
interacts with γ-aminobutyric acid (GABA)-mediated chloride channels. GABAergic system
is involved in the manifestation of sexual behavior. We previously found that IVM at
therapeutic doses did not alter sexual behavior in male rats, but at a higher dose, the
appetitive phase of sexual behavior was impaired. Thus, we investigated whether the
reduction of sexual behavior that was previously observed was a consequence of motor or
motivational deficits that are induced by IVM. Data showed significant decrease in striatal
dopaminergic system activity and lower testosterone levels but no effects on sexual
motivation or penile erection. These findings suggest IVM may activate the GABAergic
3
system and reduce testosterone levels, resulting in a reduction of motor coordination as
consequence of the inhibition of striatal dopamine release.
Keywords: Avermectin. GABA, Ivermectin, Neurotransmitters, Sexual behavior.
1. Introduction
Avermectins are broad-spectrum antiparasitic agents that are widely used in
agricultural and domestic animals [3,5,14]. In human clinical practice, avermectins are used
to treat lymphatic filariasis, onchocerciases, and other parasite infections [4,12,18].
Ivermectin (IVM) belongs to the macrocyclic lactone class of endectocides and consists of a
mixture of two homologous compounds: 22,23-dihydroavermectin B1a (H2B1a; 80%) and
22,23-dihydroavermectin B1b (H2B1b; 20%).
Avermectins exert their anthelmintic effects by binding to glutamatergic chloride
channels that are expressed on nematode neurons and pharyngeal muscle cells. Ivermectin
was the first avermectin that was synthesized. It is able to irreversibly and slowly activate
channel opening, leading to the deep and long-lasting hyperpolarization or depolarization of
neurons and muscle cells and blockade of further function [19]. In vertebrates, macrocyclic
lactones (MLs) can produce γ-aminobutyric acid (GABA)-mimetic effects by acting as
GABAA receptor agonists and stimulating GABA release [8,9,16,20]. Mammals are less
susceptible to the toxic effects of MLs because GABA-mediated nerves are found only in the
central nervous system (CNS), and MLs do not readily cross the blood-brain barrier [4,20].
However, the most common side effects of avermectins in mammals are linked to their
effects on GABAergic activation.
Sexual behavior in male rats is analyzed with regard to two distinct aspects: appetitive
phase (motivation or libido) and consummatory phase (performance). The appetitive phase is
4
characterized by the ease with which sexual behavior is activated or libido is generated. The
consummatory phase is characterized by the expression of copulatory acts. However, despite
the distinction between these stages, they are also closely related [6,23].
Previous studies from our laboratory found that doramectin [9] and IVM [17] act as
GABAergic agonists and interfere with GABAergic-related behavior, leading to anxiety-like
behavior and seizures. In female rats, doramectin and IVM impair sexual behavior during
both hormonal and natural estrus [11,15]. Furthermore, we found that IVM at a therapeutic
dose (0.2 mg/kg) did not alter sexual behavior in male rats. However, at higher doses (0.6 and
1.0 mg/kg), the appetitive phase of sexual behavior was impaired in sexually inexperienced
male rats [2]. Indeed, 0.6 and 1.0 mg/kg IVM increased the latency to the first mount and first
intromission 15 min after administration in sexually inexperienced male rats [1]. These
effects were attributed to interference with sexual motivation.
Agmo et al. [33] found no relationship between effects on locomotor activity and
sexual behavior. However, sexual behavior is inhibited whenever motor execution is
impaired. Recent studies suggest that GABA transaminase inhibitors affect sexual behavior
only indirectly through the impairment of motor execution [24,28]. Therefore, decreases in
sexual behavior may be a consequence of motor incoordination, lower motivation, or a
decrease in copulatory aspects. Thus, the present study investigated whether the reduction of
sexual behavior that was previously reported was a consequence of motor, motivational, or
erectile deficits that were induced by IVM.
Several neurotransmitters have been proposed to interfere with sexual behavior,
including motor coordination. Most pharmacological studies have focused on monoamine
neurotransmitters. For example, dopamine (DA) was suggested to stimulate aspects of sexual
behavior [13,23,28]. A relationship was found between GABA and DA in both the
5
nigrostriatal and mesolimbic DA systems. GABA and the GABA receptor agonist muscimol
inhibit the firing of DA neurons in the substantia nigra and ventral tegmental area [22,23,32].
The serotoninergic system is also involved in the manifestation of sexual behavior and
regulated by various brain areas [35]. Popova and Amstislavskaya [36] hypothesized that
serotonin 5-hydroxytryptramine-1A (5-HT1A) receptors are involved in sexual motivation in
male mice that is induced by sexually receptive females. This receptor is reported to be
primarily responsible for the stimulation and inhibition of ejaculation and penile erections
[35,37]. These authors found that the 5HT1A receptor agonist 8-OH-DPAT (0.1, 0.25, 0.5,
and 2.0 mg/kg) dose-dependently reduced the amount of time spent by male mice near female
mice, which is considered the gold standard of sexual motivation. Treatment with the 5HT1A
receptor antagonist p-MPPI (0.1, 0.2, and 0.4 mg/kg) exerted effects that were opposite to
agonists [36]. These findings suggest participation of the serotoninergic system in sexual
motivation in rodents. To reveal the mechanisms that underlie the relationship between
neurotransmitters and sexual performance, we neurochemically analyzed striatal and
hypothalamic GABA, serotonin, and DA.
2. Material and Methods
2.1. Animals
Healthy male Wistar rats (90-100 days old) from the Department of Pathology,
School of Veterinary Medicine and Animal Science, University of São Paulo (FMVZ/USP),
were used. The animals were housed by gender in groups of five in polypropylene cages with
a metal cover (40 cm × 50 cm × 20 cm) under controlled room temperature (22C ± 2°C),
humidity (45-65%), and artificial lighting (12 h/12 h light/dark cycle). Motor coordination
and penile erections were evaluated during the light cycle, and sexual motivation was
evaluated during the dark cycle.
6
The animals received free access to Nuvilab® rodent chow (Nuvital, São Paulo,
Brazil) and filtered water. Sterilized, residue-free wood shavings were used as bedding. All of
the procedures were reviewed and approved by the Animal Care Committee of FMVZ-USP
(protocol no. 2881/2013) and conformed to the guidelines of the Committee on Care and Use
of Laboratory Animal Resources, National Research Council, USA (1996). All efforts were
made to minimize animal suffering.
2.2. Drugs and treatment
Ivermectin (1% Ivomec® injectable, Merial Animal Health Ltda., Paulínia, SP, Brazil)
was dissolved in Tween 80 (1 drop/1 ml of 1% Ivomec) and administered intraperitoneally
(IP) at a dose of 0.2 or 1.0 mg/kg. Tween 80 was also administered as a control solution (1
drop/1 ml of 0.9% NaCl).
Penile erections were induced by apomorphine (Sigma, St. Louis, MO, USA).
Apomorphine was dissolved in saline solution immediately before beginning the experiments
and administered subcutaneously (SC) at a dose of 0.05 mg/kg. Moderate doses of
apomorphine (0.05-0.2 mg/kg) contribute to male sexual responses that are associated with
the genitals, such as penile erection and yawns [1,42,43].
Estrus was induced by a commercial preparation of estradiol valerate (1 mg
Primogyna®, Delpharm Lille S.A.S., imported by Bayer S.A., São Paulo, SP, Brazil) diluted
in saline solution and administered subcutaneously (SC) at a dose of 0.5 mg/kg [40]. All of
the solutions were administered in a volume of 1.0 ml/kg.
The animals were randomly assigned to three groups: 0.2 IVM, 1.0 mg/kg IVM, and
1.0 ml/kg control solution (control group). All of the behavioral observations were made 15
min after IVM administration as previously described [2] and 24 h after IVM administration
because of its half-life [25,26].
7
2.3. Procedures
2.3.1. Motor coordination
Motor coordination was evaluated using the wooden beam test as described by
Rodrigues-Alves et al. [27]. Briefly, the apparatus consisted of a wooden beam (18 mm
width, 18 mm thickness, 2 m length). Each extremity of the platform had a 10 cm  10 cm
area for the rodent. The beam was elevated at a height of 200 mm and painted white with two
black vertical marks that delimited 1 m in the middle.
Each rat was initially trained to walk on the beam in 5 min daily sessions. On the first
day, cooked corn grains (positive reinforcement) were placed on both platforms. The rat was
then introduced to the platforms to habituate them to the environment and reinforcement. The
next day, the animal was placed on the beam, close to the platform with the reinforcement,
with its head turned toward the platform. On the subsequent days, the rat was placed on the
beam at increasing distances from the platform with the reinforcement until the animal
crossed the beam, reaching the opposite platform and returning to the initial platform, always
receiving reinforcement at the end of each crossing. The training period (7-10 days) was
considered complete when the rat could reliably cross the beam without stalling (four
crossings). Few missteps were made during this stage. The animals that were unable to walk
the length of the beam within 10 days were eliminated from the experiment. Once trained,
the rats were subjected to two observation sessions (15 min and 24 h after treatment). During
each evaluation, a score was attributed for each step with the pelvic member turned to the
observer (0 = foot positioned on top of beam, no slippage; 1 = foot slip so that part of the foot
was visible below the lower surface of the beam; 2 = whole foot slip below the lower surface
of the beam) when the rat walked in the central portion of the beam at each crossing. At the
end of each session, the scores from each animal from the four crossings were summed.
8
Before each animal was tested, the wooden beam was cleaned with 5% ethanol solution. The
rats in this test were euthanized by decapitation and used to evaluate relative organ weights,
testosterone levels, and monoamine levels in the striatum and hypothalamus.
2.3.2. Sexual motivation
Sexual motivation was observed in an apparatus as described by Dahlgren et al. [7]
and Rodrigues-Alves et al. [38]. The apparatus consisted of a circular wooden arena (80 cm
diameter) that was surrounded by a 28 cm high wall. An opening (12 cm  12 cm) in the
arena wall allowed the test animals to communicate with each incentive animal cage. A wire
mesh separated the incentive animal from the experimental animal, thus allowing only visual
and olfactory contact. In front of these cages, the incentive zone (20 cm  30 cm) was
delineated in black on the arena floor. The arena floor was divided into three zones: male
incentive zone (MIZ; delineated by black lines), female incentive zone (FIZ), and the neutral
zone (NZ; defined by the remaining area of the arena). The apparatus was located in a room
that was illuminated by a red incandescent light bulb.
For sexual motivation observations, incentive animals were used. A sexually receptive
female was used for the sexual stimulation of experimental males (estrus was
pharmacologically induced by estradiol valerate 24 h before starting the experiment), and a
sexually experienced male was used for social incentive for experimental males that were
located in cages outside the arena.
The sexually inexperienced experimental rats were first habituated to the testing
environment for three 5 min sessions. The incentive animals were not present during
habituation. Immediately before each session, the arena was cleaned with 5% ethanol
solution. The test was similar to the habituation procedure and lasted 20 min, but an incentive
rat was placed in each incentive animal cage. During the test, the experimental rat could hear,
see, and smell the incentive animals, but no copulatory interactions were possible.
9
The following parameters were recorded: time (in seconds) spent in the FIZ and MIZ
and frequency of visits to each of the incentive zones (IZs; i.e., number of times that the
animal entered its four paws in the FIZ or MIZ). We also calculated the preference score,
which was the ratio of the time spent in the FIZ and total time spent in both IZs: preference
score = time in FIZ / (time in FIZ + time in MIZ).
2.3.3. Penile erection
The rats were individually placed in Plexiglas cages (30 cm × 30 cm × 30 cm) with a
mirror wall. This box was elevated 10 cm above a mirror (45 cm × 45 cm). Each rat was
simultaneously tested for penile erection and yawning that were induced by 0.05 mg/kg
apomorphine (SC).
The rats were observed for 60 min, during which time the latency (in seconds) and
frequency of spontaneous penile erections (only when the rat displayed a full erection and
bent down to lick its penis) and latency (in seconds) and frequency of yawns were recorded.
The frequency of spontaneous penile erections was defined as the number of genital reflexes,
and latency (in seconds) was defined as the time that elapsed between the injections and the
first penile erection. Yawning frequency and latency (in seconds) were recorded by visual
observations. Immediately before each session, the arena was cleaned with a 5% ethanol
solution.
2.3.4. Relative organ weight and gonadossomatic index
The rats that were previously observed for motor coordination 24 h after the
treatments were euthanized, and their brains, blood, and organs were collected. The liver,
testes, epididymis, ventral prostate, and seminal vesicle (full and empty, without the
coagulating gland) were weighed, and the relative weight (RW) was calculated: RW = (organ
10
weight / body weight)  100.
The gonadossomatic index (GSI) of male rats was calculated as the proportion of the
gonad mass relative to total body mass: GSI = (gonad weight / body weight)  100 [21]. The
GSI is used to assess the sexual maturity of testis development.
2.3.5. Histopathological examination
Representative liver fragments, the testes, and epididymis were fixed in 10%
formalin, dehydrated, diaphanized, and embedded in paraffin. The material was then cut into
5 mm thick sections on a microtome and stained with hematoxylin-eosin (HE) for
histopathological analysis.
2.3.6. Testosterone levels
Serum testosterone levels were assessed using commercially available enzyme-linked
immunosorbent assays. The procedure was performed according to the manufacturer’s
instructions (testosterone kit, Cayman Chemical, Ann Arbor, MI, USA; catalog no. 582701).
2.3.7. Monoamine levels and turnover in the striatum and hypothalamus
Hypothalamic and striatal monoamine levels, metabolite levels, and GABA levels
were evaluated by high-performance liquid chromatography (HPLC). The rats were
decapitated, and their brains were rapidly dissected on dry ice and prepared as described
previously [10]. The striatum and hypothalamus were dissected, weighed, stored at -80°C,
and used for further analysis. Following sample collection, the tissue was placed in perchloric
acid solution and homogenized by sonication for the immediate determination of
neurotransmitter and metabolite levels. Dopamine (DA) and its metabolite 3,4dihydroxyphenylacetic
acid
(DOPAC), norepinephrine (NOR)
and its
metabolite
11
vanillylmandelic acid (VMA), serotonin (5-HT) and its metabolite 5-hydroxyindolacetic acid
(5-HIAA), and GABA were measured by HPLC (Shimadzu, model 20A, Kyoto, Japan) using
a C-18 column (Shimpak, ODS, Kyoto, Japan), an electrochemical detector (Decade,
Shimadzu, Kyoto, Japan), an automatic injector (model 20A, Prominence, Shimadzu, Kyoto,
Japan), and an integrator (Chromatopac, Shimadzu, Kyoto, Japan). Each sample was run for
20 min. The detection limit for DA, DOPAC, 5-HT, 5-HIAA, and VMA was 2 pg. The
coefficients of variation were less than 15%, and the curve linearities were greater than 0.9.
Dopamine, NOR, and 5-HT turnover (metabolite/neurotransmitter ratio) was calculated.
GABA was measured by HPLC (HP, model 1100) with a Beckman 5 μ Ultrasphere
ODS-PTH column and sample injector (valve for 1.0 ml). Each sample was run for 46 min,
and the limit of detection was 20 pg.
2.4. Statistical analysis
Analysis of variance (ANOVA), followed by Dunnett’s multiple-comparison post hoc
test, was used to analyze relative organ weights, the GSI, testosterone levels, and monoamine
levels. The motor coordination, sexual motivation, and penile erection data were analyzed
using two-way ANOVA, followed by the Sidak post hoc test. The Kruskal-Wallis test was
used to analyze nonparametric data determined by the Dunn post hoc test. In all of the
experiments, p < 0.05 was the criterion for statistical significance. The data are expressed as
mean ± standard error of the mean (SEM) and median and respective minimum and
maximum limits. Instat software (Prism 6.01, GraphPad, San Diego, CA, USA) was used to
analyze the data.
3. Results
3.1. Motor coordination
12
In the motor coordination test, rats that were treated with 0.2 and 1.0 mg/kg IVM had
significantly different sums of scores (Fig. 1). The two-way ANOVA revealed significant
effects of treatment time (15 min vs. 24 h; F1,36 = 8.942, p = 0.005) and treatment (for both
IVM doses; F2,36 = 12.53, p < 0.0001) but no treatment time  treatment interaction (F2,36 =
0.5222, p = 0.5976). The Sidak post hoc test indicated a reduction of motor coordination with
both treatment times in the IVM-treated groups compared with the control group (p < 0.05).
3.2. Sexual motivation
Sexual motivation in rats that were treated with IVM was not significantly different
from the control group (Fig. 2).
3.3. Penile erection
No significant differences in penile erection were observed between the IVM-treated
groups and control group. The two-way ANOVA revealed a significant effect of group only
on the number of yawns (F2,30 = 4.580, p = 0.0184). The Sidak post hoc test revealed an
increase in the number of yawns in the group that was treated with the lower dose of IVM 15
min after administration compared with the control group (Fig. 3).
3.4. Relative organ weights and GSI
Relative organ weights and the GSI in rats 24 h after IVM administration were similar
to the control group, with the exception of the relative weight of the liver (Fig. 4). The oneway ANOVA revealed a significant effect of treatment on the relative liver weight (F2,18 =
8.407, p = 0.0026). The Dunnett post hoc test revealed an increase in the relative weight of
the liver in rats that were treated with both doses of IVM 24 h after administration compared
with the control group.
13
3.5. Histopathological examination
The histopathological examination of rats that were euthanized 24 h after treatment
did not reveal significant changes in morphology after IVM exposure (data not shown).
3.6. Testosterone levels
Treatment with IVM significantly altered testosterone levels (Fig. 5). The one-way
ANOVA revealed a decrease in testosterone levels at the highest dose of IVM 24 h after
administration compared with the control group (F2,18 = 4.372, p = 0.0283).
3.7. Monoamine levels and turnover in the hypothalamus and striatum
To examine the effects of IVM on sexual behavior and motor coordination, the levels
of neurotransmitters and their metabolites were evaluated in the hypothalamus and striatum.
As show in Table 1, in the hypothalamus, the one-way ANOVA revealed significant effects
of group on 5-HT levels (F2,15 = 12.53, p < 0.05), 5-HIAA levels (F2,16 = 16.73, p < 0.05),
and GABA levels (F2,20 = 12.15, p < 0.05). In the striatum, the one-way ANOVA revealed a
significant effect of group on 5-HT levels (F2,18 = 21.75, p < 0.05), 5-HIAA levels (F2,18 =
38.59, p < 0.05), DOPAC levels (F2,18 = 44.88, p < 0.05), and GABA levels (F2,19 = 7.43, p <
0.05). The Dunnett post hoc test revealed decreases in GABA, 5-HT, and 5-HIAA levels and
serotoninergic and dopaminergic turnover in the hypothalamus. In the striatum, 0.2 mg/kg
IVM decreased GABA, 5-HT, 5-HIAA, and DA levels and noradrenergic and dopaminergic
turnover in the hypothalamus. Ivermectin at the dose of 1.0 mg/kg increased DOPAC levels
only in the striatum.
4. Discussion
14
The present study found that 0.2 and 1.0 mg/kg IVM impaired motor coordination in
rats without influencing sexual motivation or penile erection. The impairment in motor
coordination was observed at both doses of IVM (0.2 and 1.0 mg/kg) and at both times of
observation (15 min and 24 h). These data are consistent with Agmo et al. [33], who showed
that GABAergic inhibition reduced sexual behavior by causing motor incoordination.
Similar findings were reported by Rodrigues-Alves et al. [27] using moxidectin (a
milbemycin with a similar mechanism of action as IVM). The data indicated a loss of motor
coordination in rats 24 and 72 h after administration of 0.2, 2.0, and 20 mg/kg moxidectin.
This effect was attributed to actions of the drug on the central GABAergic system, particular
GABAB receptors. GABAA and GABAB receptors can modulate locomotion and exert
opposing effects on the regulation of this behavior [27,29,30]. GABAB receptor stimulation
decreases locomotor activity, whereas GABAA receptors are not involved in this effect [31].
The present results indicated a significant decrease in the activity of the striatal dopaminergic
system suggesting that IVM may activate the GABAergic system and result in a reduction of
motor coordination through the inhibition of striatal dopamine release. Lower striatal
DOPAC levels were also observed. Therefore, the impairments in motor coordination that
were caused by IVM may be attributable to GABAB receptors and the striatal dopaminergic
system.
We evaluated whether motivational aspects were responsible for the decrease in
sexual behavior. Sexual motivation is described when a sexually inexperienced rat is exposed
to a sexual incentive (receptive female) and a social incentive (male). Male rats must choose
to remain with the sexual incentive [41]. However, no differences in sexual motivation were
observed between groups. The impairment in sexual behavior may not be attributable to
motivational aspects, and IVM does not impair the rats’ motivational state. The hypothalamus
is associated with sexual motivation and sexual behavior, and a reduction of the
15
dopaminergic system activity was observed [34]. Lower DOPAC/DA turnover was also
observed in the hypothalamus in IVM-treated rats at both doses tested, but no differences in
sexual motivation were observed despite the lower hypothalamic dopamine levels.
The effects of IVM on the consummatory phase (performance) of sexual behavior
were also assessed. Penile erection was induced by apomorphine in rats that were treated with
IVM. No differences in penile erection were observed between the control and IVM-treated
groups, indicating that IVM does not influence the consummatory phase (performance) of
sexual behavior. Neuronal activation by dopamine and dopamine receptor agonists, including
excitatory amino acids (e.g., N-methyl-D-aspartate) or oxytocin, or by electrical stimulation
leads to penile erection. The inhibition of neuronal activation by GABA, GABA receptor
agonists, opioid peptides, and opiate-like drugs inhibits this sexual response [44,45].
Ivermectin was expected to decrease penile erections that were induced by a low dose of
apomorphine once the hypothalamic dopaminergic system is reduced. However, GABA
levels in the hypothalamus increased. Thus, one possibility is that the increase in GABA
levels may modulate the decrease in hypothalamic dopamine, thus resulting in a lack of an
effect on penile erection.
We also evaluated apomorphine-induced yawning, which is a common stereotyped
behavior with unknown physiological function [42,43,47]. We observed an increase in
yawning at an IVM dose of 0.2 mg/kg 15 min after administration. The paraventricular
nucleus of the hypothalamus, also referred to as the yawning center of the brain, contains
numerous chemical messengers that can induce yawning [44,45]. A complex interaction
between DA, 5-HT, GABA, and acetylcholine is related to the expression of this behavior at
the hypothalamic level [43,46]. Ivermectin treatment had no effects on apomorphine-induced
penile erection but increased yawning, despite the fact that the same dose of apomorphine
was administered.
16
Early studies showed that serotonergic neurotransmission has an inhibitory influence
on the neural mechanisms that mediate sexual behavior [37,48]. This argument was based
mainly on observations that a reduction of brain serotonergic activity facilitates the elicitation
of sexual behavior [49], whereas an increase in central serotonergic activity may attenuate it
[50]. However, the discovery of 14 types of serotonin receptors added numerous layers of
complexity to the study of serotonin and sexual behavior. Evidence shows that certain
receptor subtypes facilitate sexual behavior [51], whereas others suppress it [50] as well as
sexual arousal and motivation. In the present study, a decrease in activity of the hypothalamic
serotoninergic system, through a decrease in 5-HIAA/5-HT, was observed at IVM doses of
0.2 and 1.0 mg/kg 24 h after administration, with no effects on penile erection or sexual
motivation. Therefore, we cannot speculate about the involvement of the IVM treatment in
the hypothalamic serotoninergic system.
We also investigated the relative weights of various organs, the GSI, and
histopathological changes. The relative weight of the liver increased in rats that were treated
with 0.2 and 1.0 mg/kg IVM, but no histopathological alterations were detected. No changes
in the GSI were observed between groups.
Sexual behavior in male rats is partially regulated by the hypothalamic-pituitarygonadal axis. Gonadotropin releasing hormone that is released from the hypothalamus
triggers the release of luteinizing hormone, which stimulates the release of testosterone from
the testes. Male rats reflexively release testosterone when they smell (anticipatory release) or
mate (ejaculatory release) with a novel receptive female [39,41]. Our results showed that 1.0
mg/kg IVM reduced serum testosterone levels, but no effects on penile erection or sexual
motivation were observed.
The endogenous male hormone testosterone and structurally related synthetic
compounds can influence behavior. Androgen significantly increases exploratory behavior
17
and motor behavior [52]. The nigrostriatal dopaminergic system plays a crucial role in
maintaining normal coordinated motor behavior [53], and decreases of the dopaminergic
activity in the nigrostriatal system promotes coordinated motor behavioral deficits.
Open field activity was previously shown to decrease in male rats that were subjected
to gonadectomy (GDX), which was rescued by androgen supplementation [53,54].
Dopaminergic activity in the brain is influenced by androgen administration [53]. The
administration of testosterone in intact male rats increased DA levels in both the neostriatum
and nucleus accumbens [55,56].
Testosterone replacement in male GDX rats restored
dopaminergic activity and modulated striatal DA storage/uptake mechanisms in GDX rats
[56,57]. In the present study, we found that lower striatal activity in the dopaminergic system
may be associated either directly or indirectly with lower testosterone levels and impairments
in motor coordination in male IVM-treated rats. We propose that the reduction of testosterone
levels may explain some aspects of motor incoordination and also may explain the previous
data that were reported by Bernardi et al. [2], in which impairments in sexual behavior were
found in male rats that were treated with the same dose of IVM (1.0 mg/kg).
5. Conclusions
The present results indicated that the decrease in male sexual behavior after IVM
administration is associated with GABA and striatal dopamine release and may also be
associated with lower testosterone levels. These systems together could modulate the
decrease in motor coordination but not sexual motivation or penile erection. Further studies
are necessary to understand the ways in which IVM influences the GABA and striatal
dopamine systems.
6. Acknowledgements
18
This work is part of the Master’s thesis presented by the first author to the School of
Veterinary Medicine and Animal Science, University of São Paulo, and was supported by
grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP; grant no. 2015/03131-4),
and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors
thank Nicolle Queiroz-Hazarbassanov for technical support.
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8. Figure Legends
Figure 1. Effects of ivermectin (0.2 or 1.0 mg/kg) and control solution (1.0 ml/kg) on motor
coordination in rats in the wooden beam test. Male rats were observed 15 min and 24 h after
administration. The data are presented as median (internal full line), first and third quartiles
(upper and lower box limits), and minimum and maximum values (upper and lower vertical
lines). N = 8 in control group, N = 6 in 0.2 mg/kg ivermectin group, N = 7 in 1.0 mg/kg
ivermectin group. C, control; 0.2, 0.2 mg/kg ivermectin; 1.0, 1.0 mg/kg ivermectin. **p <
0.01, *p < 0.05, compared with respective control group (two-way ANOVA followed by
Sidak post hoc test).
26
Figure 2. Effects of ivermectin (0.2 or 1.0 mg/kg) and control solution (1.0 ml/kg) on sexual
motivation in male rats. Sexually inexperienced rats were observed 15 min and 24 h after
administration. The data are expressed as mean ± SEM. Preference scores are reported as
median (internal full line), first and third quartiles (upper and lower box limits), and
minimum and maximum values (upper and lower vertical lines). N = 9 in control group, N =
9 in 0.2 mg/kg ivermectin group, N = 9 in 1.0 mg/kg ivermectin group. MIZ, male incentive
zone; FIZ, female incentive zone. p > 0.05, compared with control group (two-way ANOVA
followed by Sidak post hoc test).
27
Figure 3. Effects of ivermectin (0.2 or 1.0 mg/kg) and control solution (1.0 ml/kg) on penile
erections induced by apomorphine in male rats observed 15 min and 24 h after
administration. The latencies to the first penile erection are presented as median (internal full
line), first and third quartiles (upper and lower box limits), and minimum and maximum
values (upper and lower vertical lines). The data for the other parameters are expressed as
mean ± SEM. N = 6 in control group, N = 6 in 0.2 mg/kg ivermectin group, N = 6 in 1.0
mg/kg ivermectin group. **p < 0.01, compared with control group (two–way ANOVA
followed by Sidak post hoc test).
28
Figure 4. Effects of ivermectin (0.2 or 1.0 mg/kg) and control solution (1.0 ml/kg) on relative
organ weights (liver, prostate, testes, epididymis, and seminal vesicle) and gonadossomatic
index in rats 24 h after administration. The data are expressed as mean ± SEM. N = 8 in
control group, N = 6 in 0.2 mg/kg ivermectin group, N = 7 in 1.0 mg/kg ivermectin group.
**p < 0.01, compared with control group (one-way ANOVA followed by Dunnett post hoc
test).
29
Figure 5. Serum testosterone levels in male rats 24 h after administration of 0.2 or 1.0 mg/kg
ivermectin or control solution (1.0 ml/kg). The data are expressed as mean ± SEM. N = 8 in
control group, N = 6 in 0.2 mg/kg ivermectin group, N = 7 in 1.0 mg/kg ivermectin group. *p
< 0.05, compared with control group (one-way ANOVA followed by Dunnett post hoc test).
30
Table 1. Effects of ivermectin (0.2 or 1.0 mg/kg) or control solution (1.0 ml/kg) on
neurotransmitter and metabolite levels (ng/g tissue) and metabolite/neurotransmitter ratios in
the hypothalamus and striatum in male rats 24 h of administration. The data are expressed as
mean ± SEM. GABA, γ-aminobutyric acid; 5-HT, 5-hydroxytryptamine (serotonin); 5-HIAA,
5-hydroxyindoleacetic acid; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; NOR,
norepinephrine; VMA, vanillylmandelic acid; N, number of animals per group. *p < 0.05,
compared with control group (one-way ANOVA followed by Dunnett post hoc test).
Neurotransmitter
and metabolite
levels
Control
(N = 8)
Ivermectin (mg/kg)
0.2 (N = 6)
Hypothalamus
0.240±0.09*
1.0 (N = 7)
GABA
0.40±0.06
5-HT
1894.10±433.76
954.200±274.28*
1720.38±295.54
5-HIAA
2648.63±643.33
1012.57±311.10*
1895.82±502.88*
5-HIAA/5-HT
1.46±0.20
1.07±0.17*
1.11±0.26*
DA
248.79±79.59
298.55±358.33
337.26±107.51
DOPAC
65.29±23.81
69.48±62.20
59.34±30.09
DOPAC/DA
0.31±0.05
0.13±0.03*
0.18±0.09*
NOR
1226.35±254.50
1211.20±356.25
1511.18±378.99
VMA
297.09±79.97
209.67±61.45
305.18±64.72
VMA/NOR
0.29±0.11
0.18±0.06
0.21±008
0.309±0.06*
Striatum
GABA
0.42±0.06
0.32±0.02*
0.41±0.06
5-HT
936.55±204.67
373.68±140.04*
608.86±110.04*
5-HIAA
2944.86±351.76
1573.73±185.18*
2786.34±333.20
5-HIAA/5-HT
3.52±0.69
4.64±1.66
4.68±0.85
DA
5949.78±469.87
5466.13±725.62
6418.90±467.06
DOPAC
1349.15±225.59
737.92±108.34*
1786.90±223.65*
DOPAC/DA
0.24±0.04
0.14±0.01*
0.28±0.04
31
NOR
140.90±72.72
234.35±108.84
185.24±66.83
VMA
280.59±84.09
246.93±62.64
277.87±75.63
VMA/NOR
3.40±1.30
1.18±0.43*
2.91±2.41
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