Immunity and behavior: antibodies alter emotion

Abstract

Systemic lupus erythematosus is an autoimmune disease in which most patients express Abs that bind double-stranded DNA. Recent work has shown that a subset of lupus Abs can crossreact with the NR2A and NR2B subunits of the NMDA receptor. This receptor is expressed in neurons throughout the brain but is at highest density within cells of the hippocampus, amygdala, and hypothalamus. The neurons in the CNS are normally protected from brain-reactive Abs by the blood-brain barrier (BBB); however, a breach in the barrier's integrity exposes neurons to potentially pathogenic Abs. Previously, we have shown that mice that are immunized with a peptide mimetope of DNA produce lupus-like Abs that crossreact with DNA and the NMDA receptor. Moreover, after abrogation of the BBB by treatment with lipopolysaccharide, the immunized mice display hippocampal neuron damage with ensuing memory impairment. Given that rises in epinephrine can increase cerebral blood flow and can cause leaks in the BBB, we decided to investigate whether epinephrine could act as a permissive agent for Ab-mediated neurotoxicity. Here, we show that peptide-immunized mice, given epinephrine to open the BBB, lose neurons in the lateral amygdala and develop a behavioral disorder characterized by a deficient response to fear-conditioning paradigms. Thus, the agent used to open the BBB determines which brain region is made vulnerable to neurotoxic Abs, and Abs that penetrate brain tissue can cause changes not only in cognitive competence, but also in emotional behavior.

Figures

Fig. 1.

Epinephrine treatment allows selective penetration of anti-dsDNA, anti-NR2 autoAb into the amygdala. (A) Mice immunized with MAP reveal high titer of anti-peptide, anti-dsDNA Ab in the serum. Mice immunized with control MAP-core (polylysine backbone) do not. The graphs show titration of serum binding to NR2-peptide (Upper) or dsDNA (Lower). Each bar shows the mean ± SEM (n = 5 mice). Serum dilutions for each bar: 1, 500; 2, 1,000; 3, 2,000; and 4, 4,000. (B) Immunized mice with anti-dsDNA, anti-NR2 Abs show Nissl-stained amygdalar neurons that are shrunken and possess clumped nuclei (Upper). (Magnification: ×100; Inset, ×800.) These neurons also show IgG binding and are positive for Fluorojade-B, a marker of neurodegeneration. Mice immunized with MAP-core show normal amygdalar neurons with diffuse, nonneuronal IgG binding and show no evidence of neurodegeneration by Fluorojade-B (Lower). (Magnification: ×100; Inset, ×800.)

Fig. 2.

D-peptide or memantine treatment before epinephrine exposure spares amygdalar neurons in immunized mice. Anti-NR2 Abs do not destroy neurons without an epinephrine-mediated breach of the BBB. Normal neurons in the lateral amygdala of an untreated mouse (BALB/c) immunized with MAP are shown. (Magnification: ×200; Inset, ×400.) Epinephrine treatment of an animal immunized with MAP demonstrates shrunken and pycnotic amygdalar neurons. The sections are comparable, although the amygdala of an animal immunized with MAP and treated with epinephrine and D-peptide or memantine demonstrates normal neurons.

Fig. 3.

D-peptide or memantine treatment before epinephrine exposure prevents activation of caspase-3 in the amygdala of MAP-immunized mice. Neurons are identified by anti-MAP2 immunoreactivity (FITC-IgG, green). Activated caspase-3 (Rhodamine Red-X IgG, red) is apparent only in neurons from mice immunized with anti-peptide Abs and treated with epinephrine. Colocalization of MAP-2 and activated caspase-3 yields a merged yellow signal. (Magnification: ×640.)

Fig. 4.

Immunized mice that produce anti-dsDNA, anti-NR2 Abs and are treated with epinephrine show disruption in associative fear conditioning. (A) Normal motor balance in mice immunized with MAP (n = 18) and treated with epinephrine compared with MAP-core mice (n = 20). ANOVA reveals no difference between groups (F = 0.03, df = 1/31, P = 0.85). (B) Recognition memory, assessed with the two-object test, is similar between groups (discrimination ratios, t = 0.46, P = 0.65, t test). Additionally, the groups show comparable habituation to the chamber in which the two-object test will proceed. (C and D) Mice behave similarly in tests that depend on the integrity of the hippocampus: the T-maze test for spatial working memory (C)(F = 1.39, df = 1/31, P = 0.25, ANOVA on alternations) and the training-to-criterion test (D)(F = 0.92, df = 1/36, P = 0.35, ANOVA on locations). (E) Fear conditioning, assessed by fear of an auditory conditional stimulus (tone) and of the context in which the mice are conditioned, shows that the MAP group has a severely impaired fear response (freezing). The time spent freezing during the tone test is significantly lower (t = 4.31, P < 0.0005, t test), as in the context test (t = 3.1, P < 0.001, t test). All values represent the mean ± SEM.