© 2012. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which permits unrestricted non-commercial use, distribution and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the 1 same Creative Commons License terms.
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Toxoplasma gondii infection induces dendritic retraction in basolateral amygdala accompanied by reduced corticosterone secretion
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Rupshi Mitra a, Robert Morris Sapolskyb, Ajai Vyasa
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Title page
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School of Biological Sciences, Nanyang Technological University, Singapore 637551, Republic of Singapore
Department of Biology, Stanford University; Departments of Neurology and Neurological Sciences, and of Neurosurgery Stanford University Medical School, Stanford, CA 94305, USA
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Running title: Parasite induces amygdala changes.
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Number of pages: 21 Number of words in the abstract: 196 Number of words in introduction: 448 Number of words in discussion: 748 Number of figures: 5 Number of tables: 0 Number of supplemental information: 0
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Corresponding author
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Ajai Vyas
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School of Biological Sciences, Nanyang Technological University,
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Singapore 637551, Republic of Singapore
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PH: +65-6513 7365; Fax: +65-6791 3856;
[email protected]
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Acknowledgements
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This research was funded by Nanyang Technological University, Singapore; Stanley Medical Research Institute and National Institute of Health, USA (grant AI41014).
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Conflict of Interest
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Authors declare no conflict of interest.
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DMM Advance Online Articles. Posted 25 October 2012 as doi: 10.1242/dmm.009928 Access the most recent version at http://dmm.biologists.org/lookup/doi/10.1242/dmm.009928
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Abstract
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Pathological anxiety is thought to reflect a maladaptive state characterized by exaggerated
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fear. Naturally occurring perturbations that reduce fear can be crucial in the search for new
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treatments. The protozoan parasite Toxoplasma gondii invades rat brain and removes fear of
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rats for cat odors, a change believed to be parasitic manipulation of host behavior aimed at
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increasing parasite transmission. It is likely that mechanisms employed by Toxoplasma
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gondii can be used as a heuristic tool to understand possible means of fear reduction in
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clinical settings. Male Long-Evans rats were infected with Toxoplasma gondii and compared
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with sham-infected animals 8 weeks post-infection. Amount of circulating plasma
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corticosterone and dendritic arborization of basolateral amygdala principal neurons were
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quantified. Previous studies have shown that corticosterone, acting within the basolateral
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amygdala, enhances the fear response to environmental stimuli. Here we show that
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Toxoplasma gondii infection causes a dendritic retraction in basolateral amygdala neurons.
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Such dendritic retraction is accompanied by lower amount of circulating corticosterone both
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at baseline and when induced by an aversive cat odor. The concerted effects of parasitism on
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two pivotal physiological nodes of fear response provide an animal model relevant to stress
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hormones interaction with amygdalar plasticity.
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Keywords: Anxiety; Behavioral manipulation; Fear; Glucocorticoid; Hormone; Parasite
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Introduction
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Anxiety disorders are widely prevalent, have huge economic burden and are primary
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condition in co-morbid disorders (Wittchen et al., 2000; Kessler and Greenberg, 2002;
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Kessler et al., 2005; Baldwin et al., 2010). Current treatments have disappointing success rate
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and substantial side-effects (Baldwin, 2008; Baldwin et al., 2010; Ravindran and Stein, 2010).
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Hence there is a vast gap between need and availability of treatment options. Anxiety
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disorders are disorders of fear systems, reflecting exaggerated fear mismatched with actual
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environmental stimuli. Naturally occurring perturbations that reduce fear can be crucial to
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clinical search for the treatment of anxiety disorders. Behavioral effects of Toxoplasma
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gondii present such an opportunity. Toxoplasma gondii removes instinctual fear of a rat for
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cat odors, and instead generates an attraction (Berdoy et al., 2000; Webster et al., 2006; Vyas
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et al., 2007b; Vyas et al., 2007a). This change is believed to be a parasitic manipulation of
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host behavior, because of the obligatory requirement of cat intestine for parasite sexual
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reproduction. It is possible that mechanisms employed by Toxoplasma gondii to reduce fear
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can provide a heuristic tool for clinical management of excessive fear.
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Manipulations that enhance dendritic arbors or excitatory drive in the basolateral amygdala
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(BLA) concomitantly enhance anxiety (Sajdyk and Shekhar, 1997; Adamec, 1999; Vyas et al.,
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2002; Vyas et al., 2003; Rainnie et al., 2004; Adamec et al., 2005; Mitra et al., 2005; Mitra
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and Sapolsky, 2008). And manipulations that reduce these parameters concomitantly reduce
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anxiety (Mitra et al., 2009c; Mitra et al., 2009a). Similarly, exogenous application of
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corticosterone enhances anxiety (Mitra and Sapolsky, 2008) and its antagonism reduces
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anxiety (Moriceau et al., 2004; Mitra et al., 2009b). Interestingly, increases in corticosterone
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concentrations and in BLA dendritic arbors are inter-linked, a fact that is crucial for
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regulation of fear and anxiety. The BLA influences corticosterone secretion via facilitatory
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connections with hypothalamic nuclei initiating stress hormone secretion (Herman et al.,
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1996; Herman and Cullinan, 1997; Herman et al., 2003). In turn, corticosterone facilitates
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BLA plasticity via activation of corticosteroid receptors within the BLA (Mitra and Sapolsky,
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2008; Mitra et al., 2009b). Thus greater synaptic connectivity of the BLA neurons likely
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increases corticosterone secretion; and greater corticosterone secretion likely enhances
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synaptic connectivity of the BLA. In other words, BLA and corticosterone are connected via
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a positive feedback loop that creates facilitatory changes in each of them (Mitra and Sapolsky,
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2010). The diminution of this positive feedback loop is of immense clinical significance.
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In view of critical role of BLA plasticity and corticosterone, we postulated that Toxoplasma
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gondii alters both of these pivotal nodes in fear response. In this backdrop, we study effects
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of chronic Toxoplasma gondii infection in rats on dendritic arborization of BLA neurons and
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on circulating levels of corticosterone.
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Materials and methods
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Animals, parasites and infection
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Long-Evans (≈49 days old) were obtained from Charles River laboratories (Willmington,
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MA). The Stanford University administrative panel for laboratory animal care approved all
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procedures. A Prugniaud strain of Toxoplasma gondii was used. Animals were either infected
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with tachyzoites (10 X 106, i.p.) or mock-infected with sterile phosphate buffered saline. All
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experiments were conducted between 6 to 8 weeks post-infection, a period known to harbor
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chronic infection (Vyas et al., 2007b; Vyas et al., 2007a).
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Only male rats were used in the present investigation. This limitation is particularly important
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because a greater proportion of women than men suffer from clinical disorders of anxiety
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(Kessler et al., 2005), and also because behavioral effects of Toxoplasma gondii are gender-
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dependant in mice (Xiao et al., 2012).
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Morphological measurements
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Animals were decapitated under deep flurothane anesthesia. Freshly dissected brain tissues
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containing the amygdala were processed for staining individual neurons using rapid Golgi
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method (Vyas et al., 2002; Mitra et al., 2005; Mitra and Sapolsky, 2008; Mitra et al., 2009c).
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Golgi-stained BLA tissue was sectioned (120 µm thick), mounted with cover slip and used
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for morphological analysis. Custom-designed macros embedded in ‘NIH Image’ software
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(http://rsbweb.nih.gov/nih-image/) were used for morphometric analysis of digitized images.
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Endocrine measurements
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Concentration of circulating plasma corticosterone was quantified at baseline and 25 minutes
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after exposure to 2 ml of bobcat urine. For blood collection, animals were gently held inside a
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dark towel and up to 100 μl blood was collected in heparinised tubes through a tail nick.
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Blood was drawn between 10 AM to 12 noon (3 to 5 hours after start of the light-phase) when
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corticosterone is at the trough of its diurnal cycle. Time interval between picking up animal
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from home-cage and end of blood collection was less than two minutes. This method is
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known to induce minimal stress during repeated blood collection (Fluttert et al., 2000). The
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tubes were kept on ice and were subsequently centrifuged to collect the plasma (5415C,
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Eppendorf; 10000 rpm for 10 minutes). Corticosterone titers in plasma (diluted 11 times)
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were assessed using a competitive enzyme immunoassay kit (Assay Design, Minneapolis,
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MN). This assay typically results in a sensitivity value of 27 pg/ml (concentration of CORT
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two standard deviation away from zero on standard curve). This assay method has low cross-
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reactivity to testosterone (< 0.15%).
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Statistics
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Values are reported as mean ± SEM, and percentage changes are calculated with respect to
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corresponding control values (N is provided in figure legends). Two way analysis of variance
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was conducted to discern effects of infection and hemisphere on dendritic length and also
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when analyzing effects of infection and predator odor exposure on corticosterone levels.
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Independent sample student’s t-test was employed as post-hoc test. Orthogonal planned
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comparisons were used to compare effects of infection separately in left and right hemisphere
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(Student’s t-test, Bonferroni correction applied). No mean was compared more than once
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during the planned comparison.
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Results
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Toxoplasma gondii infection induced dendritic retraction in basolateral amygdala
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We quantified total dendritic length of principal neurons of the BLA eight weeks after
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infection with either Toxoplasma gondii or a sham injection of buffered saline, a time-frame
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when behavioral effects of the infection are present (Vyas et al., 2007b). We quantified left
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and right hemispheres separately.
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A two-way analysis of variance (ANOVA) revealed significant main effects of both infection
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and hemisphere. Neurons from infected animals exhibited reduced dendritic length (Figure 1a,
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30% reduction in marginal mean; F(1,152) = 37.6, p < 0.000001). Likewise, neurons sampled
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from right basolateral amygdala exhibited lower dendritic length compared to left (Figure 1b,
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15% reduction in marginal mean; F(1,152) = 7.6, p < 0.01). The effect of interaction between
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infection status and hemisphere was not statistically significant (F(1,152) = 0.1, p = 0.75)
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Planned comparisons demonstrated that infection induced dendritic retractions of 30% and
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31% in the left and right hemisphere, respectively (Figure 2; independent sample t-test; p
0.6). This shows that the retraction observed in the BLA
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does not represent a generic infection-induced atrophy of neurons.
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Toxoplasma gondii infection reduced circulating levels of corticosterone
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Effect of infection on circulating plasma corticosterone was quantified at both baseline and
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25 minutes after the presentation of two ml of bobcat urine. A two-way analysis of variance
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revealed significant effect of infection (F(1,33) = 75.5, p < 0.000001), cat odor (F(1,33) = 9.5, p
