Rats infected with the protozoan Toxoplasma gondii exhibit a reduced aversion to cat odor. This behavioral change is thought to increase trophic transmission of the parasite. Infected male rats also show a greater testicular synthesis of testosterone and epigenetic change in arginine vasopressin within the medial amygdala. Here, we show that exogenous supply of testosterone within MeA of uninfected castrates recapitulates reduction in innate fear akin to behavioral change attributed to the parasite. We also show that castration post establishment of chronic infection precludes changes in fear and medial amygdala arginine vasopressin in the infected male rats. These observations support the role of gonadal hormones and pursuant neuroendocrine changes in mediating the loss of fear in the infected rats. This work also demonstrates that testosterone acting specifically within the medial amygdala sufficiently explains reduced defensive behaviors often observed during the appetitive component of reproductive behaviors.
Introduction
Laboratory rats and mice infected with Toxoplasma gondii show reduced aversion to cat odors and an atypical attraction in a subset of animals (1, 2). This reduction in fear is assumed to increase predation by cats, who act as the definitive host for the parasite. Unequivocal evidence of greater predation for infected rats is still lacking (3). Nevertheless, loss of fear in this host-parasite relation presents a unique opportunity to study mechanisms of defensive behaviors. Several hypotheses have been advanced about proximate causation of reduced fear in this model (4–5). These possibilities can be grouped in two broad classes, including those that envision a central role of brain invasion by the parasite and those that argue that the brain invasion is merely incidental (4).
One among these hypotheses suggests that testicular invasion by Toxoplasma gondii initiates a neuroendocrine cascade (6). This invasion then leads to an increase in testosterone production and downstream epigenetic change in the medial amygdala of the brain, eventually leading to behavioral change due to the role of the medial amygdala in semiochemical processing (4, 7). Infection of rats with Toxoplasma gondii increases the amount of luteinizing hormone receptor and other rate-limiting enzymes involved in the synthesis of testosterone from its precursor in Leydig cells of the tests (6, 8). This causes an increase in circulating testosterone, which then crosses the blood-brain barrier and enhances transcription of arginine vasopressin (AVP) in the medial amygdala (7, 9). These neurons are part of the extra-hypothalamic vasopressin system and are important modulators of sociosexual behavior in male rodents (10–11). It is hypothesized that an increase in the tone of the medial amygdala vasopressin system reduces fear by increasing approach behavior (12), in line with the role of these neurons in facilitating reproductive behaviors which are often traded off with the defense. The important role of gonadal steroids is also supported by an increase in the production of major urinary proteins by male rats post-infection (13, 14), a phenotype that is testosterone-dependent (15). Moreover, Toxoplasma gondii infection increases behavioral impulsivity resembling effects produced by testosterone (16–17).
This leads to two unique predictions. Firstly, targeted testosterone supplementation within the medial amygdala should be sufficient to recapitulate behavioral change sans infection. Secondly, the removal of testes should lead to the rescue of host behavioral change. These predictions are in contrast to hypotheses built around the requirement of parasitic invasion of the brain itself. In the present report, we experimentally test these divergent predictions.
Materials and Methods
Animals
Male adult Wistar rats were used, procured from the National University of Singapore (>8 weeks at the start of the experiment). All experimental procedures were reviewed and approved by local institutional animal use and care committee. Standard laboratory animal housing conditions were employed (12:12 light-dark cycle; lights on at 07:00 h; ad libitum food and water). The number of animals was estimated based on behavioral variance observed during a previous study with similar experimental procedures. All animals survived experimental procedures until the sacrifice.
Testosterone Supplementation Experiment
Rats were treated with prophylactic antibiotic and peripherally acting analgesia 15 min prior to surgery. Surgery was performed using aseptic techniques under isoflurane anesthesia (2.5% gaseous isoflurane with pure O2). Testes and vas deferens were bilaterally removed through a medial incision in the scrotum. Bilateral intra-cerebral cannulas were also implanted dorsal to posterodorsal division of the medial amygdala (AP = −3.0, L = ± 3.8, v = −7.0) (18). Osmotic pumps were placed subcutaneously and connected thorough cannulas to supply either testosterone (25mM ethanol stock solution diluted to 3% in artificial cerebrospinal fluid) or placebo (3% ethanol solution in artificial cerebrospinal fluid) to the medial amygdala (ALZET Osmotic Pumps, USA). Body weights were recorded for seven days post-operatively, with no further experimental procedures planned during this period.
Aversion to bobcat urine was measured after 12 successive days of supplementation. Animals were first habituated to the testing arena (two rectangular arms of 76 cm × 9 cm each, connected by a central junction of 9 cm × 9 cm); for two consecutive days in the absence of odor. Subsequently, rats were placed in the arena pre-seeded with bobcat urine and rabbit urine in two opposing corners of the arena (2 ml, trial duration = 20 min).
The correct placement of the cannula was retrospectively confirmed using histological examination. Animals were sacrificed through transcardial perfusion of 4% paraformaldehyde dissolved in phosphate-buffered saline. Harvested brains were equilibrated in 30% (wt/vol) sucrose. Coronal sections through the medial amygdala were obtained at 40-μm thickness at −22°C (Leica CM1950 cryostat). Slide-mounted sections were examined at 400X after Nissl staining (19). One animal exhibiting incorrect placement in both hemispheres was excluded from statistical analysis. Five animals with incorrect placement in only one of the hemispheres were not excluded. Animals with unilateral placements within the medial amygdala in were not excluded due to lack of a priori expectation of lateralization and anatomic separation from other known androgen-responsive neuronal populations. Cannula tracks remained invisible in two animals; these animals were included in the analysis.
Castration Experiment
A type 2 Toxoplasma strain, Prugniaud, was used for the infection at a dose of 5 million lab-grown tachyzoites per animals (i.p.). Corresponding control animals were injected with sterile buffered saline. Routine management of animals and parasites was similar to earlier studies (7). All animals were castrated >6 weeks post-infection. Surgery was performed under deep anesthesia achieved by 2% isoflurane gas mixed in pure O2. A medial scrotal incision was made, testes were bilaterally removed along with vas deferens, and blood vessels supplying the testis were cauterized. The scrotal incision was closed using wound clips, which were removed after 1 week. Animals were monitored daily for 1 week, and no experimental procedures were planned during this period.
Aversion to cat odor was measured ten days post-castration. Brains were harvested by decapitation immediately after behavioral testing. Posterodorsal medial amygdala was microdissected from harvested brains and genomic DNA was isolated using DNeasy Blood and Tissue Kit (Qiagen). The methylation-sensitive restriction enzyme (HpaII, New England Biolabs, USA) digestion assay was used to quantify the level of methylation of the AVP promoter in the genomic DNA, using methods described before (7). The following primers were used to estimate DNA abundance of AVP promoter site: forward GTAGACCGCCACACCTGA and reverse CCAGACATTGGTGTGTGACC.
Data Analysis
The normality of the data was tested using the Shapiro-Wilk test (20). The assumption of normality was found to be void in case of escape latency from bobcat odor for testosterone supplementation experiments. This set of data was analyzed using unpaired Student’s t-test before and after rank transformation, with a similar outcome with respect to the probability of type 1 error. Values for non-transformed data are reported. All other endpoints were determined to normally distribute and unpaired Student’s t-test was used to calculate p values. The effect size was calculated using Cohen’s d.
Results
Exogenous Testosterone Within the Medial Amygdala Recapitulated the Behavioral Effects of the Infection
Castrated animals chronically supplemented with testosterone within the posterodorsal medial amygdala were compared with vehicle-treated animals for aversion to bobcat urine. Five out of seven vehicle-treated animals and four out of twelve testosterone-treated animals successfully escaped from the arena before completion of 1200-s-long trial. Inter-group comparison revealed that testosterone treatment significantly enhanced escape latency (Figure 1A; independent sample t-test: t17 = 2.90, p = 0.01). Analysis of the effect size demonstrated a robust difference due to testosterone treatment (mean difference: ׀∆x̅Ι = 521 ± 180 s; effect size: Cohen’s d = 1.30). Both experimental groups exhibited a significant aversion to the cat odor as demonstrated by reduced occupancy of bisect containing cat urine (Figure 1B; one-sample t-test against a chance expectation of 50%; t6 = 15.11 for placebo and t11 = 2.76 for testosterone, p < 0.02). Inter-group comparison demonstrated significant reduction in aversion to cat odor due to testosterone treatment (t17 = 2.24, p = 0.039; ׀∆x̅Ι = 16.1 ± 7.2%; Cohen’s d = 1.19). Robustness of testosterone treatment on all three endpoints measured here is borne out by the high magnitude of effect size (d > 1). Moreover, the 25th percentile of the testosterone-treated group was placed well above the median of placebo-treated animals in terms of both the number of sorties made and time spent near cat urine. Testosterone treated animals also conducted more sorties to vicinity of the cat odor (Figure 1C; t17 = 3.06, p = 0.007; ׀∆x̅Ι = 8.9 ± 2.9; Cohen’s d = 1.61).
