T-lymphocyte activation increases hypothalamic and amygdaloid expression of CRH mRNA and emotional reactivity to novelty

Abstract

Stimulation of T-cells with staphylococcal enterotoxin B (SEB) significantly elevates interleukin-2 (IL-2) and contemporaneous activation of the hypothalamic-pituitary-adrenal (HPA) axis and c-fos in the paraventricular nucleus (PVN) of BALB/cByJ mice. Such neural signaling may promote cognitive and emotional adaptation before or during infectious illness. Because corticotropin-releasing hormone (CRH) is an anxiogenic neuropeptide that may mediate the stressor-like effects of immunological stimuli, we measured neuronal CRH mRNA alterations in mice challenged with SEB. Increased CRH mRNA levels were observed in the PVN and central nucleus of the amygdala (ceA) 4-6 hr after SEB administration. This was associated with plasma ACTH increases, which could be abrogated by the systemic administration of anti-CRH antiserum. Additional experiments did not support a role for IL-2 or prostaglandin synthesis in activating the HPA axis. Behavioral experiments testing for conditioned taste aversion did not confirm that SEB challenge promotes malaise. However, consistent with the notion that central CRH alterations induced by SEB may affect emotionality (e.g., fear), SEB challenge augmented appetitive neophobia in a context-dependent manner, being marked in a novel and stressful environment. It is hypothesized that immunological stimuli generate a cascade of events that solicit integrative neural processes involved in emotional behavior. As such, these data support the contention that affective illness may be influenced by immunological processes and the production of cytokines and are consistent with other evidence demonstrating that autoimmune reactivity is associated with enhanced emotionality.

Fig. 1.

IL-2 transcription is increased in the spleens of mice 2, 4, and 6 hr after SEB challenge. Mice were challenged intraperitoneally with 50 μg of SEB at 0, 2, 4, or 6 hr before death and assessment of IL-2 mRNA by in situ hybridization of spleen tissue sections. Unchallenged mice (A) showed no detectable levels of IL-2 mRNA in the lymphocyte-rich white pulp (WP) regions of the spleen. This was not the case 2 hr (B) and 4 hr (C) after SEB challenge. Hybridization for IL-2 mRNA was dramatic at these times and confined principally to the WP regions (arrows) rather than to the macrophage-enriched red pulp (RP). The level of hybridization decreased appreciably by 6 hr (E). This is more clearly evident by comparing the images in D and F, which offer higher magnification views of silver grain density within thedotted circles in C (4 hr) andE (6 hr), respectively. Scale bars: A–C,E, 200 μm; D, F, 20 μm.

Fig. 2.

SEB increases CRH mRNA levels 6 hr after challenge in the hypothalamus (PVN) and amygdala (ceA). This figure shows representative autoradiographic film images (insets) and dark-field silver grain formation of the ceA and PVN from subsequently emulsion-dipped slides. A, ceA/control.B, ceA/SEB. C, PVN/control.D, PVN/SEB. Scale bars: A–D, Dark field, 100 μm; insets, 2 mm.

Fig. 3.

Increased CRH mRNA hybridization in the central nucleus of the amygdala 6 hr after SEB challenge. The ceA region in emulsion-dipped and nuclear-counterstained brain tissue sections were captured digitally under light-field illumination. A, Control. B, SEB. Clear evidence of more intense and numerous silver grain formation over cytoplasmic domains is evident in the SEB-challenged animal (compare the areas indicated byarrows). Scale bars: A, B, 25 μm.

Fig. 4.

Mean disintegrations per minute (DPM ± SE) in the PVN and ceA of CRH mRNA-hybridized brains from control and SEB-challenged animals. Animals were killed at the same time of day 0, 2, 4, and 6 hr after challenge with 50 μg of SEB (n = 6–8 per group). Film autoradiographs were captured digitally and quantified as described in Materials and Methods. *Significantly different from 0 hr at p < 0.05.

Fig. 5.

Effect of systemic CRH immunoneutralization and indomethacin treatment on plasma ACTH concentrations after SEB challenge. A, Animals were treated with either sheep anti-rat CRH or normal sheep serum and 15 min later were challenged intraperitoneally with 50 μg of SEB or saline. It is evident that there was complete abrogation by anti-CRH treatment of the ACTH response to SEB (n = 6–9 per group).B, Animals were treated intravenously with vehicle or indomethacin (10 mg/kg) and challenged with saline, 50 μg of SEB, or 50 ng of hIL-1β. Measurement of plasma ACTH 2 hr later revealed no alteration because of INDO treatment in the elevated ACTH concentrations observed in SEB-challenged animals. A statistically significant reduction in plasma ACTH was observed in the IL-1 group pretreated with INDO (see Results).

Fig. 6.

SEB challenge enhances the neophobic avoidance of a novel taste stimulus. Several experiments measured consumatory behavior under various conditions of environmental and food novelty (see Materials and Methods). Data represent the mean consumption ±SE (n = 6 per group, Experiment 1;n = 8 in Experiments 2 and 3; see Results for full statistical details). A, Effect of SEB challenge on consumption of a novel taste stimulus in a novel environment 2 hr after challenge, Test 1. Animals were reexposed to the drinking situation (same environment and drinking stimulus) on the next day (Test 2, i.e., day 2) and on day 6 after challenge (Test 3). Consumption levels for SEB-challenged animals were reduced significantly. B, Different groups of animals were preexposed (H, i.e., habituated) or remained naïve (N, i.e., novel environment) to the drinking test environment. Consumption 2 hr after SEB challenge was reduced significantly only if the environment was novel (Test 1, day 1). Consumption remained low on subsequent test days (D4 and D9) but displayed recovery.C, Effect of varying the novelty of the drinking stimulus. One group of animals was preexposed to the drinking stimulus in the home cage (Familiar Taste group) while another group received water (Novel Taste group). Consumption of the distinct drinking stimulus in a novel environment was measured 2 hr after SEB or saline challenge. There was no effect on consumption by SEB challenge if the drinking stimulus was familiar.