Original Paper Received: September 5, 2013 Accepted after revision: October 16, 2013 Published online: February 6, 2014

Neuroimmunomodulation 2014;21:195–205 DOI: 10.1159/000356521

Helminth Infection Alters Mood and Short-Term Memory as well as Levels of Neurotransmitters and Cytokines in the Mouse Hippocampus Jorge Morales-Montor b Ofir Picazo d Hugo Besedovsky a Romel Hernández-Bello h Lorena López-Griego b Enrique Becerril-Villanueva e Julia Moreno f, g Lenin Pavón e Karen Nava-Castro c Ignacio Camacho-Arroyo c  

 

 

 

 

a

 

 

 

 

 

Department of Immunophysiology, Institute of Physiology, Philipps-University, Marburg, Germany; b Departamento de Inmunología, Instituto de Investigaciones Biomédicas and c Facultad de Química, Departamento de Biología, Universidad Nacional Autónoma de México, d Escuela Superior de Medicina, Sección de Estudios de Posgrado e Investigación, Instituto Politécnico Nacional, e Laboratorio de Psicoinmunología de la Dirección de Investigaciones en Neurociencias and f Dirección de Servicios Clínicos, Instituto Nacional de Psiquiatría ‘Ramón de la Fuente’, and g Hospital de Ginecología No. 3, Centro Médico ‘La Raza’, Instituto Mexicano del Seguro Social, Mexico City, and h Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Mexico  

 

 

 

 

 

 

 

Abstract Helminthic infections are important causes of morbidity and mortality in many developing countries, where children bear the greatest health burden. The ability of parasites to cause behavioral changes in the host has been observed in a variety of host-parasite systems, including the Taenia crassicepsmouse model. In murine cysticercosis, mice exhibit a disruption in the sexual, aggressive and avoidance predator behaviors. Objective: The present study was conducted to characterize short-term memory and depression-like behavior, as well as levels of neurotransmitters and cytokines in the hippocampus of cysticercotic male and female mice. Methods: Cytokines were detected by RT-PCR and neurotransmitters were quantified by HPLC. Results: Chronic cysticercosis infection induced a decrease in short-term memory in both male and female mice, having a more pronounced effect in

© 2014 S. Karger AG, Basel 1021–7401/14/0214–0195$39.50/0 E-Mail [email protected] www.karger.com/nim

females. Infected females showed a significant increase in forced swimming tests with a decrease in immobility. In contrast, male mice showed an increment in total activity and ambulation tests. Serotonin levels decreased by 30% in the hippocampus of infected females whereas noradrenaline levels significantly increased in infected males. The hippocampal expression of IL-4 increased in infected female mice, but decreased in infected male mice. Conclusion: Our study suggests that intraperitoneal chronic infection with cysticerci in mice leads to persistent deficits in tasks dependent on the animal’s hippocampal function. Our findings are a first approach to elucidating the role of the neuroimmune network in controlling short-term memory and mood in T. crassiceps-infected mice. © 2014 S. Karger AG, Basel

Introduction

Helminthic infections are an important cause of morbidity and mortality in both developing and developed countries. Overall, in developing countries, children (8– Jorge Morales Montor Circuito Interior S/N Ciudad Universitaria México D.F. 04510 (Mexico) E-Mail jmontor66 @ biomedicas.unam.mx

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Key Words Short-term memory · Neurotransmitters · Neurocytokines · Helminthes · Cysticercosis · Taenia crassiceps · Hippocampus · Immunity · Infection

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Neuroimmunomodulation 2014;21:195–205 DOI: 10.1159/000356521

tion of the estrous cycle [20]. Regarding social behavior, infection disrupts the dominant-subordinate status [21]. Infected animals assume a subordinate status as compared with healthy ones [22]. Cytokines and chemokines have been reported as modulators of brain development, regeneration and synapses [16, 23–25]. In the brain of mammals, neurons predominantly communicate through chemical synapses mediated by numerous neurotransmitters. Neurotransmitters could also modulate cytokine secretion by brain tissue. For instance, 5-HT is a neurotransmitter and immune modulator necessary for the synthesis of IL-6 and TNF-α in the brain. On the other hand, extracellular 5-HT concentrations above the baseline physiological levels may suppress the production of the above cytokines [26]. To our knowledge, no studies examining the effects of parasite infections on short-term memory, mood and neurotransmission have been performed. Since intraperitoneal infection with the helminth parasite T. crassiceps has been shown to alter different behaviors in the host, together with an alteration in the expression of several genes in specific areas of the brain [27, 28], we hypothesized that infection may also affect other aspects of the host brain physiology, such as short-term memory and mood, and that these behaviors may be related to changes in the levels of neurotransmitters and cytokines in the hippocampus. Thus, the aim of this study was to investigate changes in short-term memory, mood, neurotransmitters and cytokine expression levels in the male and female mice hippocampus during T. crassiceps infection, and to correlate these data with the parasite burdens in these animals. Our results indicate that the intraperitoneal cysticercotic infection modifies the expression pattern of cytokines and neurotransmitters within the hippocampus, which is involved in controlling short-term memory behavior.

Materials and Methods Ethics Statement The Animal Care and Use Committee at the Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM) evaluated animal care and experimentation practices according to the official Mexican regulations (NOM-062-ZOO-1999), which are in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH and The Weatherall Report) of the USA, to ensure compliance with established international regulations and guidelines. The Ethics Committee of the Instituto de Investigaciones Biomédicas approved protocol (Permission No.: 2009-16).

Morales-Montor  et al.  

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12 years old) bear the greatest health burden due to helminthic infections [1, 2]. The most important morbidities associated with helminthic infection in children are undernutrition, anemia, low physical growth and poor cognitive performance, among others [3–5]. In brief, an association has been described between helminthic infection and children’s academic performance or cognitive function, in which there is an improvement after the administration of an antihelminthic treatment [6]. In a variety of host-parasite experimental systems, behavioral changes such as exploration, total and sexual activity, and aggression have also been observed in the host. These changes are considered to be adaptive, since they ensure the transmission of parasites between hosts, and/or enhance the probability that parasites can be released in an appropriate location [7, 8]. In trophic transmission, the parasite often modifies the exploratory behavior, activity, aggression, and possibly sexual attraction of the intermediate host, which renders it more vulnerable to be preyed on by the final host [9]. For example, rodents infected with Toxoplasma gondii show a diminished fear of the parasite’s definitive host – the cat [10–12]. The relationship between parasites and their hosts implies a biochemical coevolution that involves a tight communication between complex physiological and metabolic systems at different levels. In many cases, parasites are thought to secrete mimetic molecules [13], or to induce the production of hormones and neurotransmitters such as dopamine (DA), noradrenaline (NA), serotonin (5-HT), opioids, γ-aminobutyric acid, glutamate, vasopressin, oxytocin, glucocorticoids, sex steroids and nitric oxide, among others, in the host [9]. There are a myriad of molecules that are thought to act on the central nervous system (CNS) of the host, inducing an altered behavior [9, 13, 14]. Murine intraperitoneal cysticercosis is caused by the helminth cestode taenid Taenia crassiceps, and has contributed to the complexity of the interactive network that regulates infection [15, 16]. Briefly, a feminization process occurs in chronically infected male mice, whose serum estradiol levels progressively increase until reaching those observed in females; whereas testosterone levels drop to 10–15% of the normal male levels [17–19]. This drop is associated to changes in both sexual and social behaviors. Sexual behavioral changes include complete loss of the ejaculation response and a gradual decrease in the number of mounts and intromissions with an increase in their latencies [18]. In female mice, infection also causes perturbations in sexual behavior, characterized by a reduced receptivity to the male, as well disrup-

 

 

Object Recognition Task The object recognition task is a nonspatial and hippocampaldependent memory test, which is indeed considered as a useful tool for evaluating the ability of rodents to evocate short-term events [30]. This test was conducted, with minor modifications, according to previous descriptions [31, 32]. The task consists of two sessions; an acquisition trial and a test trial, which takes place 1 h later. During the acquisition trial, each mouse was placed into an open box (40 × 40 × 30 cm; dim light with an intensity of 12 lx) containing two identical objects located in adjacent corners of this open field. Mice were allowed to freely explore both objects for 10 min. Exploration was scored only when the mouse’s nose or front paws touched the objects. Before the test trail, all objects used were carefully washed with a 10% ethanol solution and one of the objects was replaced by a new object. Objects were placed in the same locations as in the first trial. The mouse was returned to the arena and the total time spent in exploration of each object was determined. In this session, the total time spent exploring each object (N = novel; F = familiar) during 5 min was registered as the mean of two stopwatches. Data were calculated considering the time spent exploring both objects during the first 3 min of the test trial using the formula [(N – F)/(N + F)] × 100, and were expressed as a recognition index. In this test, a reduction in exploration of the novel object is considered as a measurement of clear impairment of short-term memory [31]. Forced Swimming Test This procedure was carried out following a modified version of the conditions proposed by Porsolt et al. [33, 34]. Each mouse was introduced into a Plexiglass cylinder (height 25 cm; diameter 10 cm) containing 15 cm of water at 25 ° C. Two swimming sessions were conducted: an initial 15-min pretest followed by a 5-min test, 24 h later. Only the second session was videotaped for later scoring. After swimming, each animal was dried, warmed and returned to its home cage. According to Detke et al. [35], a time-sampling technique was employed to score three different mice behaviors at the  

 

Helminth Infection Alters Memory in the Host

end of each 5-second period during the test session (5 min): (1) immobility (floating in the water and only doing those movements necessary to keep the head above the water); (2) swimming (showing active swimming motions, more than those necessary to keep the head above water, i.e. moving around in the cylinder or diving), and (3) climbing (presenting active movements with the forepaws in and out of the water, usually directed against the walls) [35]. Spontaneous Activity A further test was implemented to control false results in both the object recognition and forced swimming tests. All animals were evaluated in an automatic activity counter. In this test each mouse was placed in an acrylic cage (51.1 × 9.5 × 69.2 cm) with two arrays of 15 infrared-emitting beams placed perpendicular to each other. Beams were spaced 2.5 cm apart, in such a way that the interruption of each beam generated an electric impulse, which was presented as a count (Opto-Varimex; Columbus Instruments, Columbus, Ohio, USA). Total activity, ambulation and vertical activity (rearing) were registered over a 5-min test period. Neurotransmitter Measurements Brains of uninfected and infected male and female mice were immediately excised after euthanasia. The hippocampus was excised according to The Mouse Brain in Stereotaxic Coordinates [36]. Briefly, the hippocampus was obtained by cutting with a razor blade underneath the frontal cortex and placed in a 1.5-ml microcentrifuge tube on dry ice. Ice-chilled 0.1 M PCA containing internal standard was added to the tissue tube (10 mg tissue in 300 μl PCA). Samples were briefly sonicated with a microprobe, fitting in the sample tube (6–7 s, duty cycle 80%, output control) until the tissue was completely homogenized. Tubes were kept on crushed ice or at 4 ° C for 10 min. Samples were then centrifuged at 14,000 rpm. (18,000 g) for 15 min, at 4 ° C. The supernatant was transferred into another clean 1.5-ml microcentrifuge tube and centrifuged again as above. Pellets were washed and used to determine protein concentrations. Supernatants were used to determine neurotransmitter levels: NA, adrenaline (AD), DA and 5-HT for HPLC assay. Specific standards to each peak in the mixture by its retention time and voltammetric response were added. Ten microliters of all the standards and samples were filtered through a 0.45-μm filter and injected into the column. Neurotransmitter concentrations were determined by reversed-phase chromatographic analysis, using a Waters Spherisorb ODS2 C18 column, (80 Å, 5 μm, 4.6 × 250 mm) installed in a chromatographic system integrated of Jasco PU-2085 pump and AS-2057 autosampler, with an Antelec Leyden Decade II electrochemical detector, controlled by Millennium 32 software. The separation of analytes was performed at 30 ° C with a mobile phase consisting of 5% of acetronile in a buffer solution [12.16 mM citric acid, 11.60 mM (NH4)2HPO4, 2.34 mM sodium octylsulphonate, 3.32 mM dibutyl phosphate amine and 1.11 mM sodium EDTA] at isocratic conditions with a flow rate of 1 ml/min and detected in the next acquisition conditions: range 1 nA, filter 0.005 Hz, Eox 0.60 V, basal 0.001 V, Ic 2.72 nA. The order and retention time of the eluted monoamines and their metabolites in the chromatogram were: NA (5 min), AD (6 min), DA and 5-HT (18.5 min).  

 

 

 

 

 

Hippocampus Cytokine Expression Total RNA was isolated from the hippocampus of control and infected mice of both sexes by the extraction method using TRIzol reagent (Gibco-BRL, Grand Island, N.Y., USA). Briefly, each tissue was

Neuroimmunomodulation 2014;21:195–205 DOI: 10.1159/000356521

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Animals and Experimental Infections Male and female Balb/c AnN (H2-d) inbred mice obtained from Harlan (Mexico City, Mexico) were used in all of the experiments. Animals were housed in the animal care facilities at the Instituto de Investigaciones Biomédicas (UNAM), under controlled temperature conditions (22 ° C) and 12-hour dark-light cycles with lights on between 07:00 and 19:00 h. They were fed Purina Diet 5015 (Purina, St. Louis, Mo., USA) and tap water ad libitum. Estrous was evaluated in females. The fast-growing ORF strain of T. crassiceps [29] was used for infection in all of the experiments. Larvae were obtained from 3- to 6-month-infected female donor mice. Ten nonbudding T. crassiceps larvae (approximately 2 mm in diameter) were suspended in 0.3 ml of sterile phosphate-buffered saline (0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.2) and intraperitoneally injected into 42-day-old male and female mice using a 0.25 gauge needle. Ten noninfected mice of each sex were used as age-matched controls. Infected and control mice were housed in separated cages (5 each) in the same room of the animal facility. At 16 weeks of infection, control and infected mice were used to test behavioral parameters, and 1 day after the tests were performed mice were rapidly euthanized by cervical dislocation after anesthesia with pentobarbital (Pfizer, Mexico City, Mexico), always at 08:00 h. All tissue sections were immediately collected after rinsing.

Table 1. Oligonucleotide sequences used for cytokine expression

Name (size)

Forward sequence

Reverse sequence

IL-1β (503) IL-4 (181) IL-6 (638) TNF-α (300) IFN-γ (247) 18S (219)

5′-tcatgggatgatgatgataacctgct 5′-cgaagaacaccacagagagtgagct 5′-acctggtagaagtgatgccccaggca 5′-ggcaggtctactttggagtcattgc 5′-agcggctgactgaactcagattgtag 5′-cgcggttctattttgttggt

5′-cccatactttaggaagacacggatt 5′-gactcattcatggtgcgacttatcg 5′-ctatgcagttgatgaagatgtcaaa 5′-acattcgaggctccagtgaattcgg 5′-gtcacagttttcagctgtataggg 5′-agtcggcatcgtttatggtc

Primers were designed based on sequenced mouse genes from the Gene Databank (NCBI, NIH).

removed and immediately disrupted in TRIzol reagent (1 ml/0.1 g tissue) at low speed in a Brinkman Polytron Homogenizer (0.2 ml of chloroform was added per ml of TRIzol). The aqueous phase was recovered after 10 min of centrifugation at 14,000 g. RNA was precipitated with isopropyl alcohol, washed with 75% ethanol and redissolved in RNase-free water. The RNA concentration was determined by absorbance at 260 nm and its purity was verified after electrophoresis in 1.0% denaturing agarose gel in the presence of 2.2 M formaldehyde. Total RNA from all of the extracted tissues was reverse transcribed followed by specific PCR amplification of the IL-1β, IL-4, IL-6, TNF-α, IFNγ, and 18S gene sequences. Nucleotide sequences of the primers used for amplification are shown in table 1. Briefly, 1 μg of total RNA from each tissue was incubated at 37 ° C for 1 h with 400 units of M-MLV reverse transcriptase (Applied Biosystems, Boston, Mass., USA) in 20 μl of reaction volume containing 50 mM of each dNTP and 0.05 μg of oligo (dT) primer (Gibco-BRL). Ten microliters of the cDNA reaction were subjected to PCR, the 50 μl of the reaction included 10 μl of previously synthesized cDNA, 5 μl of 10× PCR buffer (Biotecnologías Universitarias, Mexico City, Mexico) 1 mM MgCl, 0.2 mM of each dNTP, 0.05 μM of each primer and 2.5 units of Taq DNA polymerase (Biotecnologías Universitarias). After an initial denaturation step at 95 ° C for 5 min, for all genes, temperature cycling was as follows: to amplify IL-1β, 95 ° C for 30 s, 57 ° C for 45 s and 72 ° C for 45 s during 35 cycles; for IL-4, 90 ° C for 30 s, 60 ° C for 45 s and 72 ° C for 50 s during 30 cycles; for IL-6, 95 ° C for 45 s, 60 ° C for 55 s and 72 ° C for 50 s during 30 cycles; for TNF-α, 90 ° C for 50 s, 57 ° C for 45 s and 72 ° C for 30 s during 40 cycles; for IFN-γ, 95 ° C for 30 s, 58 ° C for 35 s and 72 ° C for 40 s during 35 cycles. An extra extension step was completed at 72 ° C for 10 min for each gene. The 50 μl of the PCR reaction were electrophoresed on 2% agarose gel in the presence of a 100-bp ladder as a molecular weight marker (GibcoBRL). The PCR products obtained were visualized by staining with ethidium bromide. In all cases, different PCR conditions were assessed until a single band corresponding to the expected molecular weight of the gene was obtained. In order to determine if all genes amplified as well as the constitutively expressed control gene, 18S were in the exponential phase of amplification, and to be sure that changes in expression were not artifactual (such as 18S being in the stationary phase), we performed RNA quantity, cycling and temperature curves for each analyzed gene. Gel ethidium bromide staining generated signals, were quantified by densitometric scanning and are represented as the ratio of the signal from the problem gene (cytokines) relative to the expression of 18S, a constitutively expressed gene used as the internal control (relative expression).  

 

 

 

 

 

 

 

 

 

 

 

 

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Neuroimmunomodulation 2014;21:195–205 DOI: 10.1159/000356521

Number of Parasites The individual number of parasites recovered from infected mice of both sexes, as expected, showed a clear sexassociated susceptibility. Female mice reached a parasite number of 1,952.3 ± 44.6 (mean ± SD) per mouse at 16 weeks of infection, while male mice showed half the parasite burden compared to females (985.6 ± 66.0; p < 0.001). Parasites were exclusively found in the peritoneal cavity of infected mice, but not in any other organ or host compartment such as the brain. Object Recognition Test Figure 1 shows data from the object recognition test (short-term memory). It is clear that infection significantly reduces the percentage of time spent exploring the new object [F(3, 26) = 25.40; p < 0.05] in a sex-indepenMorales-Montor  et al.  

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Experimental Design and Statistical Analysis A multifactorial analysis of variance (ANOVA) was applied in order to find statistical differences. The independent variables were infection (two levels: yes or no) and gender (two levels: male or female). The dependent variables were the number of parasites, the levels of each cytokine (IL-1β, IL-4, IL-6, IFN-γ and TNF-α) in the hippocampus, as measured by the optical density of the corresponding gel divided by the optical density of 18S in the same tissue sample in the same gel, used as a control gene for PCR, and the levels of neurotransmitters in each brain region. Two experimental repetitions including tissues from 5 normal or infected mice were included in the analysis. A statistical analysis of variance components was performed with the software Prism 2.01 (GraphPad Software Inc., La Jolla, Calif., USA). When applied, post hoc individual contrasts of group means by the Tukey test used the sum of residual and three-factor interactions variance to test for significant differences. Behavioral analysis was done by means of a two-way ANOVA taking into account the infection as one factor and the gender of mice as another. Similarly to biochemical data, Tukey’s test was applied for paired comparisons.

Control Infected

Recognition (%)

80 60 40

*

*

20 0

Females

Males

Fig. 1. Effect of the infection with T. crassiceps of male and female

Balb/c AnN mice on memory performance using the object recognition test. A multifactorial ANOVA was applied in order to find statistical differences. Bars represent mean ± SD of 7–8 animals in each group. Tukey’s test; * p < 0.05 versus the respective control group.

dent fashion, since both male and female mice were sensitive to the consequences of infection. However, infected females presented an increased reduction in exploration (57%) as compared with infected males (40%). Total Activity, Ambulation and Vertical Activity Regarding total motor activity, it was observed that infected males showed a significant increase in total activity and ambulation while, in contrast, infected female mice showed no significant modifications in these parameters (fig. 2a, b). Vertical activity (fig. 2c) was not influenced by chronic infection, sex or the interaction of these factors.

of AD related to sex or infection (fig. 4c). ND (not detected) was indicated when we were unable to measure the levels of any neurotransmitter. Cytokine Expression Figure 5 shows the relative expression of IL-4, IL-1β, IL-6, TNF-α and IFN-γ in the hippocampus of infected mice. For all cytokines, a single band was detected. In order to determine if genes amplified as well as 18S were in the exponential phase of amplification, and to ensure that changes in expression were not artifacts (such as 18S being in the stationary phase), we performed RNA, cycling and temperature curves for each analyzed gene. Infection increased the IL-4 mRNA levels in female mice but induced a significant reduction in male mice (fig. 5a). There were no changes in the relative expression of IL-1β in the hippocampus of infected mice of both sexes, compared to uninfected animals (fig.  5b). Interestingly, there was a 4-fold increase in the expression of IFN-γ observed in infected males when compared to control mice (fig.  5c). Figure 5 also depicts the expression pattern of two proinflammatory cytokines (TNF-α and IL-6). TNF-α expression in the hippocampus also showed a significant difference between infected and control males and females at 16 weeks of infection (2-fold increase; fig. 5d) while the expression of IL-6 showed a 1-fold increase in the hippocampus of both infected female and male mice (fig. 5e).

Discussion

Neurotransmitter Levels Figure 4 shows that DA and 5-HT levels in the hippocampus of control female mice were higher than those in control males (fig.  4a, b). Interestingly, infection produced a clear decrease of 5-HT levels in female mice (fig. 4b) together with a moderate increase of NA in males (fig. 4d; p < 0.05). We did not find differences in the levels

In this study, we have shown an important neuroimmunoendocrine interaction in male and female mice infected with T. crassiceps cysticerci. Helminthic infections are an important cause of morbidity and mortality in many developing countries, in which children bear the greatest health burden. The ability of parasites to cause behavioral changes in the host has been observed in a variety of host-parasite systems, including the T. crassiceps mouse model. This experimental setting is a fascinating example of the interactive host-parasite neuroimmunoendocrine network. It has been shown that hormonal changes in cysticercotic mice profoundly affect their behavior, including sexual activity [18], aggressiveness [37], social status and defense response [21]. Male mice infected with T. crassiceps show remarkable changes in sexual behavior characterized by a complete loss of the ejaculation response early in the infection (6 weeks), followed by a gradual decrease in the number of mounts and intromissions, as well as increasing in their latencies, until

Helminth Infection Alters Memory in the Host

Neuroimmunomodulation 2014;21:195–205 DOI: 10.1159/000356521

Forced Swimming Test In the forced swimming task, infected female mice showed a decrease in immobility (fig. 3a), while infected male mice showed only a nonsignificant reduction. This decrease in immobility was attributable to an increase in swimming behavior (fig. 3b). In contrast, climbing was not modified by the infection (fig. 3c). Male infected mice did not exhibit significant changes in this test.

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100

200 0

Females

Males

*

400 200 0

b

Females

a

12

Number of counts

Number of counts

Fig. 2. Spontaneous activity of male and female Balb/c AnN mice chronically infected with T. crassiceps. a Total activity. b Ambulation. c Vertical activity. Behavioral analysis was done by means of a two-way ANOVA taking into consideration the infection as one

10 8 6

*

4 2 0

Females

Males

b

100 50 0

c

Females

6

10 8

*

6 4 2 0

150

Females

female Balb/c AnN mice submitted to the forced swimming test. Behavioral analysis was done by means of a two-way ANOVA taking into consideration the infection as one factor and the gender of mice as another. Data are presented as mean of counts ± SD of

none of the parasitized mice showed any sexual response toward female mice [18]. Moreover, it was demonstrated that changes in sexual behavior were due to the change in the normal production of sex steroids, since testosterone or dihydrotestosterone restitution of the infected male mice generated a complete restoration of their sexual behavior [18]. Considering that c-fos and progesterone receptor are key estradiol-regulated genes involved in the regulation of sexual behavior, we previously studied possible changes in the expression of these genes in the CNS of infected male mice. Indeed, both c-fos [38] and progesterone receptor expression [28] oscillated with time of infection and to different magnitudes in the hypothalamus, brain cortex and preoptic area, but not in other areas of the brain or in several other organs of the host. Among the helminthes, Toxocara canis is the one that induces effects on behavior and cognition, mainly because the parasite can establish in the brain and induces Neuroimmunomodulation 2014;21:195–205 DOI: 10.1159/000356521

Males

factor and the gender of mice as another. Each bar represents the mean ± SD of the number of beam interruptions for 5 min. Tukey’s test; * p < 0.05 versus the respective control group. n = 10 for male and female control mice, and for male and female infected mice.

12

Fig. 3. Effect of the chronic infection with T. crassiceps on male and

200

Males

Control Infected

Males

c

Control Infected

5 4 3 2 1 0

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Males

immobility (a), swimming (b) or climbing (c) behavior registered every 5 s during a 5-min test period. Tukey’s test; * p < 0.5 versus the corresponding uninfected group. n = 10 for male and female control mice, and for male and female infected mice.

behavioral changes in the host [39]. In 2001, Holland and Cox [39] demonstrated impairment in learning ability and exploration by using a ‘T’ maze and water-finding task in mice and rats infected with T. canis larvae. They found that infected mice were less explorative and less responsive to novelty in the ‘T’ maze, and this effect was more pronounced in heavily infected mice. These data are very consistent with our present findings in which chronically infected mice have a pronounced decline in memory tasks. The evolutionary implication of these results could be related with the fact that the parasite impairs the normal exploration of the host facilitating in this way predation and, thus, an enhanced transmission of the parasite to the definitive host in the wild [39]. Hamilton et al. [40], in 2006, reported that infected BALB/c mice with T. canis, following a period of deprivation, had a significantly increased latency in water drinking compared to control mice, indicating some degree of memory impairMorales-Montor  et al.  

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400

600

Number of counts

600

800

Number of counts

*

Number of counts

Number of counts

800

a

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1,000

1,000

30 20 10 0

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4

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**

2

6 4 2

ND

c

*

8

6

0

*

60

10

NA (pmol/10 μl)

AD (pmol/10 μl)

8

*

ND Females

Males

d

0

Females

Males

Fig. 4. a–d Neurotransmitter levels in the hippocampus of control and T. crassiceps-infected age-matched male and female mice. Results of neurotransmitter levels are reported as the pool of all mice hippocampi from each group. ND indicates that values were below the control standard used in the HPLC test. A multifactorial ANOVA was applied in order to detect statistical differences. Sta-

tistical analysis of variance components was performed with the software Prism 2.01 (GraphPad Software Inc.). When applied, post hoc individual contrasts of group means by Tukey’s test used the sum of residual and three-factor interactions variance to test for significant differences. Values are the mean ± SD. * p < 0.05, ** p < 0.01 with respect to control (uninfected age-matched) mice.

ment. Spatial awareness and the ability to use visual cues from the surrounding environment to remember locations of specific resources are vital to the survival of small rodents. Authors relate this effect in infected mice (having higher latencies to enter the alcove and drink from the water bottle) to lethargy, since parasitic infection can render a subject less energetic than uninfected individuals. Infected mice could have been more sluggish than uninfected mice, taking them longer to complete any of the tasks set in the experiment. Another important contribution from this study was the fact that they tried out to correlate the parasite burden in the brain and its location with the behavioral changes observed in the mice. The evidence of memory impairment in T. canis-infected mice in this study is of particular interest since it was evident that heavier cerebral larval burdens lead to more pronounced behavioral alterations [40]. Previous studies have reported the presence of larvae of T. canis in the telencephalon [41] and cerebellum [42] of infected mice,

which are the areas of the brain associated with learning and memory, and the coordination and control of voluntary movement. Chieffi et al. [43], in 2010, also demonstrated behavioral changes induced by T. canis infection. In this study, Rattus norvegicus were infected with T. canis larvae. Thirty and 60 days postinfection, infected and control rats were exposed to an open-field maze for 5 min and, subsequently, to an elevated plus maze, also for 5  min. An increase in exploratory behavior in infected rats was found. These results provide some support to the hypothesis that the infection in rats induces a higher exposure to the predator (dogs) in order to enhance the transmission rate of this parasite. The improvement in mobility and exploratory behavior are features in the host that could readily enhance the transmission rate of T. canis by predation of rodent paratenic hosts [43]. It is interesting to note that using a different helminth parasite, Strongyloides ratti, in rats and mice, Braithwaite et al. [44] did not find evidence of an effect of infection on learning

Helminth Infection Alters Memory in the Host

Neuroimmunomodulation 2014;21:195–205 DOI: 10.1159/000356521

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a

80

Control Infected

**

5-HT (pmol/10 μl)

DA (pmol/10 μl)

40

a

2.0

***

1.5

***

1.0 0.5 0

Females

Males

IL-1DŽ relative expression

IL-4 relative expression

2.0

b

c

***

1.5 1.0 0.5 0

Females

Males

1.0 0.5 0

***

***

Females

Males

d

Females

***

Males

***

1.5 1.0 0.5 0

Females

Males

2.0

1.0 0.5 0

Neuroimmunomodulation 2014;21:195–205 DOI: 10.1159/000356521

infected males, whose immobility values did not change (fig. 3a). Other researchers have reported similar inconsistencies about the role of 5-HT in learning and depressive regulation. For instance, Carlini et al. [49] found that adult male mice chronically treated with the selective 5-HT reuptake inhibitor fluoxetine presented an increase in 5-HT levels, but showed a poor memory retention in the object recognition test. Deleterious actions in memory, after chronic treatment with fluoxetine, have also been reported at clinical levels [50], specifically on visual memory [51]. Previously, we have reported high values of estradiol in female mice infected with T. crassiceps cysticerci [17] that corresponds with a rise in gonadotropins folliclestimulating hormone, FSH, and luteinizing hormone, LH [52]. In accordance with this observation, Carretti et al. [53] found that high gonadotropin levels correlate well with low tryptophan levels and, contrarily, when LH and FSH levels are low, tryptophan levels increase. Thus, Morales-Montor  et al.  

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e

1.5

or memory ability. Furthermore, rodents that are infected with T. gondii show an increased exploratory behavior, activity, aggression and possibly sexual attraction, which makes them more conspicuous and less afraid of the definitive host – the cat (Felis catus) [10–12]. Two of the most compromised neurotransmitters involved in learning and depression-like behavior events are NA and 5-HT [45–48]. It has been proposed that in the forced swimming test a reduction in immobility (an antidepressant effect) reflects an activation of the serotonergic system when accompanied by an increase in swimming behavior, whereas the increase in climbing behavior is considered as an index of noradrenergic activation [35]. Thus, reduction of immobility produced by infection in female mice (an antidepressant effect) should be explained by an increase in the hippocampal levels of 5HT, since such action was the consequence of swimming. However, 5-HT was almost absent in infected female mice, whereas NA in this group was not higher than in 202

1.5

2.0 TNF-į relative expression

pus of control and T. crassiceps-infected mice. Data are presented as the mean ± SD of two different experiments (n = 5). A multifactorial ANOVA was applied in order to detect statistical differences. Statistical analysis of variance components was performed with the software Prism 2.01 (GraphPad Software Inc.). When applied, post hoc individual contrasts of group means by Tukey’s test used the sum of residual and three-factor interactions variance to test for significant differences. Each cytokine was quantified in triplicate after a 16-week infection period. *** p < 0.001; compared to the control group.

IL-6 relative expression

Fig. 5. Effect of chronic infection in the expression of IL-4 (a), IL-1β (b), IFN-γ (c), TNF-α (d) and IL-6 (e) in the hippocam-

IFN-Dž relative expression

2.0

Control Infected

low 5-HT levels observed in infected females are in line with this biochemical evidence but do not agree with behavioral actions. Although this discrepancy may be attributed to differences in the experimental models used here, as well as other methodological factors (doses, treatment and species, among others), current data suggest that changes in short-term memory and depression after a chronic infection with cysticerci should be regulated by other molecules different to NA and 5-HT. Supporting this idea, the depletion of allopregnanolone, a reduced metabolite of progesterone, produces depressive behaviors [54, 55] whereas some substances with glutamatergic properties seem to have antidepressant effects [56, 57]. It is also important to consider that the discrepancies found in neurotransmitter levels and behavioral changes in our experimental setting may be methodological, since we measured the levels of the different neurotransmitters in whole hippocampus tissue, and we did not consider the extracellular neurotransmitter release, which may be the key to explain our findings in this regard. More experiments are necessary to explain the current observations and to demonstrate this hypothesis. Chronic stress favors the establishment of mental illness in individuals with a genetic background of susceptibility [58]. There are reports that describe depression-like behaviors in mice infected with the protozoan parasite Trypanosoma cruzi [59]. However, at this moment, the molecular mechanisms involved in this phenomenon have not been elucidated. Depression-like behaviors induced in mice by T. cruzi infection have been mainly related to neuroendocrine immune variations [60]. Previously, variations in the levels of cytokines like IL-1, TNF-α and IL-6 [60, 61], as well their expression in the brain, have been reported [59], although the impact of sex in these differences has not been studied. We also found changes in cytokine expression in the brain of male and female mice experimentally infected in the peritoneal cavity with T. crassiceps cysticerci. Our data indicate that changes in cytokine expression in the hippocampus of the infected mice point to the host’s CNS sensing the presence of the parasites, even if they are located in the peritoneal cavity. We evaluate IL-1β, IL-4, IL-6, TNF-α and IFN-γ, because they are typical pleiotropic cytokines that modulate a variety of physiological events in vertebrates, such as cell proliferation, differentiation, survival, apoptosis and behavior. These cytokines play roles in the immune, the endocrine, the nervous and the hematopoietic systems, and on bone metabolism. Many immune cell types are reported to produce IL-1β, IL-4, IL-6, TNF-α

and IFN-γ, including T and B cells, polymorphonuclear cells, eosinophils, monocyte/macrophages, mast cells and dendritic cells. Other cell types known to produce them are chondrocytes, osteoblasts, endothelial cells, skeletal and smooth muscle cells, islet cells, thyroid cells, fibroblasts, mesangial cells, keratinocytes, certain tumor cells, adipose tissue cells, microglial cells and astrocytes. Regarding human infections, it has been reported that in Tanzania and China, schistosomiasis infection reduces cognitive performance [62, 63]. Jukes et al. [63] and Nokes et al. [62] found that high-intensity infection with S. haematobium was associated with cognitive impairment in verbal and short-term memory domains among school-age children in Tanzania. Ezeamama et al. [64] demonstrated that children infected with Trichuris had almost 4.5-fold greater odds of performing poorly in tests of verbal fluency regardless of infection intensity. Our study provides strong evidence of association between a helminthic infection and memory in male and female mice. To our knowledge, this is the first study demonstrating a relationship between helminth infection and short-term memory. We demonstrated that T. crassiceps infection is associated with a poor performance in the object recognition test. Our finding concerning the decrease in performance during short-term memory tasks in mice may be congruent with data in humans living in endemic areas for helminthic infections, in which infected children may have a learning disadvantage relative to uninfected children [63]. Finally, our results indicate that parasite infection modifies the expression pattern of cytokines and neurotransmitters within the hippocampus, which is involved in controlling short-term memory.

Helminth Infection Alters Memory in the Host

Neuroimmunomodulation 2014;21:195–205 DOI: 10.1159/000356521

Acknowledgments Financial support was provided by grant IN-214011-3 from Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT), Dirección General de Asuntos del Personal Académico (DGAPA), Universidad Nacional Autónoma de México (UNAM) and grant 176803 from Fondos de Investigaciones Sectoriales, CB-SEP Consejo Nacional de Ciencia y Tecnología (CONACyT), México, both to J. Morales-Montor; and by grants from COFAA and SIP; INPRF NC092318.0 and NC 092318.1 to L. Pavón. Karen Nava-Castro has a postdoctoral fellowship from DGAPA, UNAM.

Disclosure Statement

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The authors declare that no conflicts of interest exist.

References

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