Figure 4.

Intraperitoneal (ip) injection of CASPR2 immunoglobulin G (IgG) with lipopolysaccharide (LPS) causes behavioral and neuropathological changes in mice. (A) Experimental design and selected behavioral tests. The behavioral tasks assessed locomotion (open field, OF), strength (inverted screen, IS), coordination (accelerating rotarod, AR; and narrow beam, NB), working memory (continuous spontaneous alternation, CSA), short- (forced alternation, FA) and long-term memory (novel object recognition, NOR—NORf, familiarization phase; NORt, test phase), anxiety (light-dark box, LDb), compulsive-like behavior (marble burying test, MB), social behavior (reciprocal social interaction tests, RSI), and olfaction (olfaction test, OT). (B) Continuous spontaneous alternations were reduced in CASPR2-IgG-injected mice compared with HC-IgG-injected mice (P = 0.044). In the RSI test, there was reduced latency to interact (P = 0.04; Mann–Whitney test) but no differences in the interaction time or number of interactions. However, in the non-social aspects of the test, there was increased time spent immobile (U = 0.008), reduced rearing (U = 0.02), and reduced grooming (U = 0.018). (C) Bound human IgG in the hippocampi and cerebellum of CASPR2- and HC-IgG-injected mice. CASPR2-IgG-injected animals had higher levels of IgG in the cortex (Cx) (P = 0.03), hippocampus (Hip) (P = 0.023), and thalamus (Th) (P = 0.0004) compared with HC-IgG-injected mice. No differences were observed in the levels of CASPR2 expression in the same areas (n = 4 per group). (D) c-Fos expression in the entorhinal–piriform cortex (P = 0.020), dorsomedial hypothalamus (DMH) (P = 0.037), and lateral hypothalamus (LH) (P = 0.031) was higher in the CASPR2-IgG-injected mice than in the HC-IgG-injected mice (n = 4 per group). (E) Representative images of glial fibrillary acidic protein (GFAP) staining in the molecular layer of the cerebellum and quantification of the mean fluorescence intensity in the same area showing higher GFAP expression in the CASPR2-IgG-injected mice (P = 0.043) (n = 4 per group; 40X, 10 μm). On the right, representative images of complement C3 expression on GFAP-positive cells. Percentage of C3/GFAP area ratio per cell showed increased C3 expression of astrocytes in CASPR2-IgG-injected mice. (F) Representative images of the z-stack projected IBA1 staining used for morphological analysis (40X, 10 μm). Quantification of morphological data in the hippocampus and molecular layer of the cerebellum showed that microglia from CASPR2-IgG-injected mice had a higher cell soma/cell total body size ratio [t₍₆₎ = 4.74, P = 0.0032] and shorter [t₍₆₎ = 3.68] ramifications than HC-IgG-injected mice, compatible with an activated phenotype in both the hippocampus (P = 0.017 and P = 0.010, respectively) and the cerebellum (P = 0.0003 and P = 0.008, respectively). ^***The contents of this figure are taken from Giannoccaro et al. (51) with permission from Oxford University Press. ^* < 0.05, ^** ≤ 0.01, ^*** ≤ 0.001.