Campylobacter jejuni infection increases anxiety-like behavior in the holeboard: Possible anatomical substrates for viscerosensory modulation of exploratory behavior

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Abstract

The presence of certain bacteria in the gastrointestinal tract influences behavior and brain function. For example, challenge with live Campylobacter jejuni (C. jejuni), a common food-born pathogen, reduces exploration of open arms of the plus maze, consistent with anxiety-like behavior, and activates brain regions associated with autonomic function, likely via a vagal pathway. As yet, however, little is known regarding the interface of immune sensory signals with brain substrates that mediate changes in behavioral states. To address this issue, we challenged mice with either C. jejuni or saline, and 7–8 h later assessed anxiety-like behavior using the open holeboard, and used immunohistochemical detection of the protein c-Fos as an activation marker in the brain. C. jejuni treatment was associated with increased avoidance of the center regions of the holeboard, compared to saline-treated controls. Exposure to the holeboard induced activation in multiple brain regions previously implicated in anxiety-like behavior, including the lateral septum (LS), paraventricular (PVN) and dorsomedial hypothalamic nuclei (DMH), basolateral and central nuclei of the amygdala (BLA, CEA), bed nucleus of the stria terminalis (BST) and periaquiductal grey (PAG), compared to homecage controls. In C. jejuni-treated animals c-Fos induction also occurred in autonomic regions, as previously reported. The PVN, BLA, parts of the BST, medial prefrontal (mPFC) and anterior cingulate responded to both C. jejuni treatment and the holeboard, suggesting a role for these regions in the enhanced anxiety-like behavior observed. In saline-treated animals, anxiety-like behavior was predicted by activation in the CEA and BLA, whereas in C. jejuni-treated animals, c-Fos expression in the BST predicted the degree of anxiety-like behavior. These findings implicate the PVN, amygdala and BST as interfaces between gastrointestinal pathogenic challenge and brain regions that mediate behavioral responses to stress, and reinforce these nuclei as anatomical substrates by which viscerosensory stimuli can influence behavior.

Introduction

Oral administration of certain bacteria, including the common food-borne bacterial pathogen Campylobacter jejuni (C. jejuni) enhances anxiety-like behavior in mice (Lyte et al., 1998, Lyte et al., 2006). Although this local gastrointestinal infection does not provoke evidence of a systemic immune response in these mice, it induces c-Fos expression (an activation marker) in vagal sensory neurons that likely innervate the gut in the first few hours following administration (Goehler et al., 2005, Lyte et al., 2006). Thus, vagal sensory pathways likely provide a major conduit by which this gastrointestinal infection can influence behavior. Consistent with this idea, infection with C. jejuni increased c-Fos expression in several brain regions that include the nucleus of the solitary tract (NTS), the lateral parabrachial nucleus (LPB), central amygdala (CEA) and the hypothalamic paraventricular nucleus (PVN), the bed nucleus of the stria terminalis (BST), and the medial prefrontal cortex (mPFC; Gaykema et al., 2004, Goehler et al., 2005). These brain regions comprise prominent components of the network of brain regions that process visceral/autonomic information, including those that are typically activated following vagal stimulation (Naritoku et al., 1995).

Although it is becoming increasingly evident that viscerosensory information regarding states such as peripheral arousal, pain, satiety, or inflammation can modulate ongoing behavior and mood (e.g., Craig, 2002, Goldstein and Silverman, 2006, Miyashita and Williams, 2006), as yet the neural substrates that allow this interaction are not established. “Bottom-up” viscerosensory (systemic/interoceptive) challenges with e.g., satiety hormones (Rinaman, 2003, Viltart et al., 2006), glucoprivation (Ritter et al., 2001) or immune challenge (Gaykema et al., 2007), activate ascending neural projections from the caudal brainstem NTS and ventrolateral medulla (VLM) that target a constellation of brain regions associated with the autonomic network, including the brainstem nuclei locus coeruleus (LC), LPB, and periaquiductal grey (PAG) as well as forebrain regions including the PVN and CEA (e.g., Billig et al., 2001, Gaykema et al., 2004, Goehler et al., 2005, Elmquist et al., 1993, Elmquist and Saper, 1996, Wan et al., 1993). This pattern overlaps with brain regions previously implicated in mediation of fear and anxiety (e.g., hypothalamus, amygdala and BST; Walker et al., 2003). Because these areas seem to serve as integrators of autonomic responses to stimuli signaling exteroceptive threats (Herman et al., 2003), they constitute key candidates for an interface between viscerosensory signals and affective states.

The previous studies investigating neuronal responses (c-Fos induction) to bacterial infection assessed animals resting their home cages, and thus reported only the c-Fos response to infection. Because c-Fos induction was not assessed following exposure to a novel environment, or any other potentiall anxiogenic situation, it was not possible to determine any relationship between c-Fos expression in the brain and anxiety-like behavior. Therefore, the present study aimed at identifying brain regions that may serve to integrate exteroceptive and interoceptive challenges (e.g., novel environment and infection) and potentially mediate the effects of immune-related viscerosensory modulation of anxiety-like behavior. We inoculated mice with C. jejuni orally and evaluated exploratory behavior using the holeboard (HB). This apparatus is designed to provide a novel, potentially dangerous environment, in which inhibition of exploration of the center, the most exposed part, is taken as an index of anxiety-like behavior. Induction of c-Fos protein in the brain was used as a neuronal activity marker. Behavior and associated brain c-Fos expression patterns of mice following oral administration of either saline or C. jejuni were compared to identify potential areas that may mediate the effects of intestinal infection on anxiety-like behavior. Based on the established overlap of viscerosensory and stress-responsive functions, we expected that a convergence of signals relevant to anxiety-like behavior and C. jejuni treatment would be revealed via c-Fos expression in the amygdala (CEA and BLA), BST and PVN. In addition, other brain regions implicated in viscerosensory processing and anxiety-like behavior were assessed as well.

Section snippets

Animals

Thirty-eight 5-week-old CF-1 male mice were purchased from Charles River Laboratories (Wilmington, DE). Upon arrival, the mice were housed one animal per cage (width: 16 cm, length 22 cm, and height 13 cm; made of opaque polypropylene) and placed in a 12-hour light/dark cycle (dark: 21:00-09:00 h, light: 09:00-21:00 h). Food and water were available ad libitum. The last cage and bedding changes were done at least 48 h preceding experiments. Prior to testing, the animals were acclimated to

C. jejuni increases anxiety-like behavior in the holeboard

Although both the saline and C. jejuni-treated mice spent most of the time in the periphery zone of the holeboard, the avoidance of the 20 × 20 cm center zone was more pronounced in C. jejuni-treated animals (Fig. 1a). The infected mice traversed a reduced distance in the center zone (111 ± 27 cm) as compared to the saline-treated mice (231 ± 39 cm; main treatment effect F1, 20 = 6.48, p = 0.02), and entered the center zone less frequently (7.5 ± 1.7 vs. 13.3 ± 2.5 entries, F1, 20 = 3.68, p = 0.07). Infected mice

Discussion

The results from this study show that challenge with the bacterial pathogen C. jejuni increased anxiety-like responses in the open field/holeboard exploration task compared to saline treated controls, (as expected based on previous studies) in that infected mice displayed a reduced willingness to explore the center of the holeboard. This difference in exploratory style may follow from induction of c-Fos protein in the CEA, the BLA, the BST, and the PVN, which responded to both the exploration

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