Behavioral and neural effects of intra-striatal infusion of anti-streptococcal antibodies in rats

Abstract

Group A β-hemolytic streptococcal (GAS) infection is associated with a spectrum of neuropsychiatric disorders. The leading hypothesis regarding this association proposes that a GAS infection induces the production of auto-antibodies, which cross-react with neuronal determinants in the brain through the process of molecular mimicry. We have recently shown that exposure of rats to GAS antigen leads to the production of anti-neuronal antibodies concomitant with the development of behavioral alterations. The present study tested the causal role of the antibodies by assessing the behavior of naïve rats following passive transfer of purified antibodies from GAS-exposed rats. Immunoglobulin G (IgG) purified from the sera of GAS-exposed rats was infused directly into the striatum of naïve rats over a 21-day period. Their behavior in the induced-grooming, marble burying, food manipulation and beam walking assays was compared to that of naïve rats infused with IgG purified from adjuvant-exposed rats as well as of naïve rats. The pattern of in vivo antibody deposition in rat brain was evaluated using immunofluorescence and colocalization. Infusion of IgG from GAS-exposed rats to naïve rats led to behavioral and motor alterations partially mimicking those seen in GAS-exposed rats. IgG from GAS-exposed rats reacted with D1 and D2 dopamine receptors and 5HT-2A and 5HT-2C serotonin receptors in vitro. In vivo, IgG deposits in the striatum of infused rats colocalized with specific brain proteins such as dopamine receptors, the serotonin transporter and other neuronal proteins. Our results demonstrate the potential pathogenic role of autoantibodies produced following exposure to GAS in the induction of behavioral and motor alterations, and support a causal role for autoantibodies in GAS-related neuropsychiatric disorders.

Keywords: Animal model; Dopamine; Pediatric autoimmune neuropsychiatric disorders associated with streptococcus (PANDAS); Serotonin; Streptococcus group A (GAS); Sydenham’s chorea (SC).

Figure 1

Time line and behavioral and immunological procedures included in the study assessing the effects of GAS-exposure (upper half) and in the study assessing the effects of IgG infusion (lower half). 5HT-2AR=5HT-2A serotonin receptor; 5HT-2CR=5HT-2C serotonin receptor; D1R=D1 dopamine receptor; D2R=D2 dopamine receptor; GFAP=Glial fibrillary acidic protein; NeuN=neuronal nuclei; SERT=serotonin transporter; WB=western blot.

Figure 2

Effects of streptococcal exposure on (A) food manipulation, (B) beam walking, and (C) grooming. (A) The mean and standard error (SE) of food manipulation scores of GAS (n=12) and control rats (n=16). (B) The mean and SE of the time spent on the wide and narrow beams of GAS (n=12) and control rats (n=16). (C) The mean and SE of the duration of grooming in three sessions of induced-grooming of GAS (n=9) and control (n=16) rats. *p<0.01, **p<0.0001

Figure 3

Effects of group A streptococcal (GAS) exposure on immunoreactivity of rat serum (IgG) with GAS antigen, dopamine receptors and serotonin receptors in the ELISA (A–E) and Western blot (F–I). ELISA results for anti-GAS serum IgG reactivity with: (A) GAS mutanolysin extracted antigen (GAS, n=12; control, n=10), (B) D1 dopamine receptor (GAS, n=11; control, n=10), (C) D2 dopamine receptor (GAS, n=13; control, n=10), (D) 5HT-2A serotonin receptor (GAS, n=12; control, n=10), and (E) 5HT-2C serotonin receptor (GAS, n=12; control, n=10), of sera taken from GAS and control rats, in comparison to reactivity with BSA. Western blot of pooled sera (IgG) reactivity from GAS-exposed rats compared with sera (IgG) from adjuvant-exposed control rats with: (F) D1 dopamine receptor, (G) D2 dopamine receptor, (H) 5HT-2A serotonin receptor, and (I) 5HT-2C serotonin receptor. D1-PC = anti-D1 receptor positive control; D2-PC = anti-D2 receptor positive control; 5HT-2A-PC = anti-5HT-2A receptor positive control; 5HT-2C-PC = anti-5HT-2C receptor positive control. *p<0.01, **p<0.005, ***p<0.0001

Figure 4

Effects of passive transfer of IgG from GAS-exposed and control rats to the striatum of naïve rats on (A) food manipulation, (B) beam walking, (C) grooming, and (D) marble burying. (A) The mean and standard error (SE) of food manipulation scores of rats infused with IgG extracted from GAS-exposed rats (GAS-I group, n=8), rats infused with IgG extracted from control rats (Control-I group, n=6) and naïve rats (n=8). (B) The mean and SE of the time spent on the wide and narrow beams of GAS-I rats (n=8), Control-I rats (n=6) and naïve rats (n=8). (C) The mean and SE of the duration of induced-grooming on three sessions of GAS-I rats (n=8), Control-I rats (n=5) and naïve rats (n=8). (d) The mean and SE of the number of marbles buried by GAS-I rats (n=8), control rats (n=5) and naïve rats (n=8). *p<0.05, **p<0.01

Figure 5

Representative immunofluorescence images showing IgG deposition in the striatum of a GAS-I rat (A, D, G), Control-I rat (B, E, H) and naïve rat (C, F, I). (A–C) Labeling in the dorso-lateral striatum (DLS, the injection site); (D–F) Labeling in the dorso-medial striatum (DMS); (G–I) Labeling in the ventral striatum (VS). Tissue sections were incubated with anti-rat alexa antibody 488 for visualization of IgG deposition; Blue signal indicates nuclear counterstaining (DAPI). 10 × microscope objective; scale bar=200μm.

Figure 6

Representative immunofluorescence images taken from the dorso-lateral striatum (DLS, the injection site) of a GAS-I rat (A–B, E–F, I–J, M–N, Q–R), and a control-I rat (C–D, G–H, K–L, O–P, S–T) showing labeling with markers for neurons (NeuN, A–D), D1 dopamine receptor (E–H), D2 dopamine receptor (I–L), serotonin transporter (SERT, M–P), and astrocytes (GFAP, Q–T), and colocalization of these markers with IgG deposition (B, D, F, H, J, LN, P, R, T). Tissue sections were incubated with anti-rat alexa antibody 488 for visualization of IgG deposition; IgG against NeuN was labeled with anti-mouse alexa antibody 594; D1 and D2 dopamine receptors, SERT and GFAP were labeled with anti-rabbit alexa antibody 594. Blue signal indicates nuclear counterstaining (DAPI). 20 × microscope objective; scale bar=100μm. The arrows point to colocalization between the infused IgG and the relevant marker.

Figure 7

Representative immunofluorescence images showing IgG deposition in the striatum of a GAS-I rat (A–C), Control-I rat (D–F) and naïve rat (G–I) and double labeling with a marker for neurons (NeuN). Figures A, D and G display labeling of rat IgG; Figures B, E and H display NeuN labeling; Figures C, F and I display colocalization of the rats IgG and NeuN. Tissue sections were incubated with anti-rat alexa antibody 488 for visualization of IgG deposition; IgG against NeuN was labeled with anti-mouse alexa antibody 594. Blue signal indicates nuclear counterstaining (DAPI). 40 × microscope objective; scale bar=25μm. The arrows point to colocalization between the infused IgG and NeuN.

Figure 8

Representative immunofluorescence images showing IgG deposition in the striatum of a GAS-I rat (A–C), Control-I rat (D–F) and naïve rat (G–I) and double labeling with anti-D1 dopamine receptor antibody. Figures A, D and G display labeling of rat IgG; Figures B, E and H display labeling of the D1 dopamine receptor; Figures C, F and I display colocalization of the rats IgG and the D1 dopamine receptor. Tissue sections were incubated with anti-rat alexa antibody 488 for visualization of IgG deposition; IgG against the D1 dopamine receptor was labeled with anti-rabbit alexa antibody 594. Blue signal indicates nuclear counterstaining (DAPI). 40 × microscope objective; scale bar=25μm. The arrows point to colocalization between the infused IgG and D1.

Figure 9

Representative immunofluorescence images showing IgG deposition in the striatum of a GAS-I rat (A–C), Control-I rat (D–F) and naïve rat (G–I) and double labeling with anti-D2 dopamine receptor antibody. Figures A, D and G display labeling of rat IgG; Figures B, E and H display labeling of the D2 dopamine receptor; Figures C, F and I display colocalization of the rats IgG and the D2 dopamine receptor. Tissue sections were incubated with anti-rat alexa antibody 488 for visualization of IgG deposition; IgG against the D2 dopamine receptor was labeled with anti-rabbit alexa antibody 594. Blue signal indicates nuclear counterstaining (DAPI). 40 × microscope objective; scale bar=25μm. The arrows point to colocalization between the infused IgG and D2.

Figure 10

Representative immunofluorescence images showing IgG deposition in the striatum of a GAS-I rat (A–C), Control-I rat (D–F) and naïve rat (G–I) and double labeling with anti-serotonin transporter (SERT) antibody. Figures A, D and G display labeling of rat IgG; Figures B, E and H display labeling of SERT; Figures C, F and I display colocalization of the rats IgG and SERT. Tissue sections were incubated with anti-rat alexa antibody 488 for visualization of IgG deposition; IgG against the SERT was labeled with anti-rabbit alexa antibody 594. Blue signal indicates nuclear counterstaining (DAPI). 40 × microscope objective; scale bar=25μm. The arrows point to colocalization between the infused IgG and SERT.

Figure 11

Representative immunofluorescence images showing IgG deposition in the striatum of a GAS-I rat (A–C), Control-I rat (D–F) and naïve rat (G–I) and double labeling with a marker for astrocytes (GFAP). Figures A, D and G display labeling of rat IgG; Figures B, E and H display GFAP labeling; Figures C, F and I display colocalization of the rats IgG and GFAP. Figures Tissue sections were incubated with anti-rat alexa antibody 488 for visualization of IgG deposition; IgG against GFAP was labeled with anti-rabbit alexa antibody 594. Blue signal indicates nuclear counterstaining (DAPI). 40 × microscope objective; scale bar=25μm.