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

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 7-8h 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.

Figures

Fig. 1

Infection with Campylobacter jejuni increases anxiety-like behavior during exploration in the holeboard. A. Reduced exploration by C. jejuni-treated mice (the darkly shaded bars) of the 20×20 cm center area (or zone) is reflected by decreased distance traveled within, entries into, and time spent inside the center zone, all in comparison to the vehicle-treated mice (open bars). B. Infected mice also engaged in diminished exploration of the very middle 5×5 cm zone as shown by reduced distance traveled, fewer partial entries (or pokes into) and full entries into the very middle zone. C. Infected mice made fewer visits and pokes into the mostly exposed center and moderately exposed side holes than the uninfected ones, but poked into the most protected corner holes as frequently as the saline-treated mice. D. There were no differences between the C. jejuni- and the saline-treated groups in general locomotor activity as reflected in the total distance traveled in the entire holeboard and in the distance traveled in the peripheral zone of the holeboard, indicating the absence of sickness behavior. * p < 0.05, ** p < 0.005, m: marginal significance, p < 0.086.

Fig. 2

A. Dramatic increases in the number of cells displaying c-Fos expression in selected brain areas in response to exposure to the holeboard (significant main effect of behavioral test, but not main treatment, effect in two-way ANOVA). In comparison to the home cage condition, the holeboard test leads to strong increases in c-Fos-positive cells in the medial prefrontal cortex (ILC, PLC, and ACC), the lateral septum, the medial (m) portion of the BST (ventromedial and dorsomedial parts combined), the hypothalamus (AHA, DMH, and PH), and the PAG (in ventrolateral, lateral, and dorsomedial quadrants. In addition, a smaller, but significant, increase in c-Fos expression due to C. jejuni infection was noticeable among the home cage controls in the medial prefrontal cortex (ILC, PLC, and ACC). B. Increases in the number of c-Fos-positive cells in selected brain regions primarily in response to C. jejuni challenge (significant main treatment, but not main test condition, effect in two-way ANOVA). Brain regions include the NTS, PBL, LC, and the ventrolateral portion of the BST. In addition, the NTS and BSTvl showed a holeboard exposure-related increase in c-Fos expression relative to the home cage condition, which was only apparent in the saline-treated groups. C. Increase in the number of c-Fos-immunoreactive cells in response to both C. jejuni challenge and holeboard exposure (both significant main treatment and test condition effects in two-way ANOVA) was evident in select brain regions, including the dorsolateral BST, CEA, BLA, and the PVN. Only in the CEA, the two-way ANOVA revealed a significant interaction (p < 0.05), as there was no further increase in c-Fos expression as a result of holeboard exposure in the infected mice beyond what was induced by the bacterial challenge. C. jejuni challenge vs. saline treatment: * p < 0.05, ** p < 0.005. Holeboard vs. home cage condition: # p < 0.05, ## p < 0.005, ### p < 0.0005.

Fig. 3

A-F. Photomicrographs showing increased c-Fos staining in the LS (A,B), DMH (C,D), and PH (E,F) in mice exposed to the holeboard (HB, in B,D,F) in comparison to the home cage controls (home, in A,C,E). No effects of C. jejuni challenge were observed in these regions in either home cage or holeboard groups (not shown). G-R. Infection with C. jejuni increased c-Fos staining in brainstem regions, including the NTS (G-J), LC (K-N), and PBL (O-R). This effect of infection (panels labeled C. jej in H,J [NTS], L,N [LC], and P,R [PBL]) was seen in both home cage (panels labeled “home”) and holeboard (HB) groups when compared with the saline-treated groups (panels labeled “saline” in G,I [NTS], K,M [LC], and O,Q [PBL]). Inserts depicting brain section diagrams show the locations of the photomicrographs (modified after Paxinos and Franklin, 2001). Abbreviations: 3V: third ventricle; 10: dorsal motor nucleus of the vagus; Arc: arcuate nucleus; AP: area postrema; CB: cerebellum; C.jej: C. jenuni-challenged. DMH: dorsomedial hypothalamus; LC: locus coeruleus; LDT: laterodorsal tegmental nucleus; LS: lateral septum; LV lateral ventricle; me5: mesencephalic trigeminal tract; MS: medial septum; NTS: nucleus of the solitary tract; PBL: lateral parabrachial nucleus; scp: superior cerebellar peduncle; VMH: ventromedial hypothalamus; vsc: ventral spinocerebellar tract. Scale bars: 250 μm.

Fig. 4

Photomicrographs of the medial prefrontal cortex (A-D), PVN (E-H), and the amygdala (I-L) showing increase in c-Fos-immunoreactive cells in response to C. jejuni challenge as well as holeboard exposure. For each brain region, the four panels depict each of the four experimental conditions indicated in the bottom left of the panels. Inserts depicting diagrams of brain sections show the locations of the photomicrographs (modified after Paxinos and Franklin, 2001). Abbreviations: 3V: third ventricle; ac: anterior commisure; ACC: anterior cingulate cortex; AHA: anterior hypothalamic area; BLA: basolateral nucleus of the amygdala; BST: bed nucleus of the stria terminalis; CEA: central nucleus of the amygdala; f: fornix; ILC: infralimbic cortex; LA: lateral nucleus of the amygdala; LV: lateral ventricle; PLC: prelimbic cortex; PVN: paraventricular nucleus of the hypothalamus. Scale bars in A, E, I: 250 μm, and apply to other corresponding panels.

Fig. 5

Photomicrographs of the bed nucleus of the stria terminalis (A-D), showing increase in c-Fos-immunoreactive cells in response to C. jejuni challenge (predominantly in the dorso- and ventrolateral parts) and to holeboard exposure. Each panel depicts one of the four experimental conditions indicated in the bottom left of the panels. Insert depicting a diagram of a brain section shows the location of the photomicrographs (modified after Paxinos and Franklin, 2001). Abbreviations: ac: anterior commissure; BST: bed nucleus of the stria terminalis, dl: dorsolateral part, vl: ventrolateral part; m: medial part; C. jej: treated with C. jejuni; CPu: caudate putamen; f: fornix; HB: holeboard exposure: LSV: lateral septal nucleus, ventral part. Scale bar in A: 250 μm, applies to all panels.

Fig. 6

Summary diagram of brain regions that are activated primarily in response to C. jejuni challenge (brainstem nuclei in dark grey), in response to behavioral challenge (holeboard exposure, brain regions in light grey), or both (brain regions in black). The latter regions likely represent the interface of visceral and exteroceptive input through which behavioral responses to environmental challenge, such as exposure to the holeboard, may be modified by viscerosensory challenge.