Behavioral, pharmacological, and immunological abnormalities after streptococcal exposure: a novel rat model of Sydenham chorea and related neuropsychiatric disorders

Abstract

Group A streptococcal (GAS) infections and autoimmunity are associated with the onset of a spectrum of neuropsychiatric disorders in children, with the prototypical disorder being Sydenham chorea (SC). Our aim was to develop an animal model that resembled the behavioral, pharmacological, and immunological abnormalities of SC and other streptococcal-related neuropsychiatric disorders. Male Lewis rats exposed to GAS antigen exhibited motor symptoms (impaired food manipulation and beam walking) and compulsive behavior (increased induced-grooming). These symptoms were alleviated by the D2 blocker haloperidol and the selective serotonin reuptake inhibitor paroxetine, respectively, drugs that are used to treat motor symptoms and compulsions in streptococcal-related neuropsychiatric disorders. Streptococcal exposure resulted in antibody deposition in the striatum, thalamus, and frontal cortex, and concomitant alterations in dopamine and glutamate levels in cortex and basal ganglia, consistent with the known pathophysiology of SC and related neuropsychiatric disorders. Autoantibodies (IgG) of GAS rats reacted with tubulin and caused elevated calcium/calmodulin-dependent protein kinase II signaling in SK-N-SH neuronal cells, as previously found with sera from SC and related neuropsychiatric disorders. Our new animal model translates directly to human disease and led us to discover autoantibodies targeted against dopamine D1 and D2 receptors in the rat model as well as in SC and other streptococcal-related neuropsychiatric disorders.

Figure 1

Effects of streptococcal exposure on (a) food manipulation, (b) beam walking, (c) grooming, and (d) activity. (a) Food manipulation was impaired in GAS-exposed rats. The mean and SE of food manipulation score of GAS rats (n=19) and control rats (n=16). (b) Beam walking was impaired in GAS-exposed rats on the narrow beam only. The mean and SE of the time spent on the wide and narrow beams of GAS rats (n=9) and control rats (n=10). (c) Induced-grooming was enhanced by GAS exposure. The mean and SE of the duration of induced-grooming on three sessions of GAS (n=8) and control (n=8) rats. (d) Activity tended to increase following GAS exposure. The mean and SE of activity counts, in 5 min blocks, of GAS (n=20) and control (n=17) rats during a 30-min activity session. ^*Significantly different from the control group, P<0.05.

Figure 2

Effect of (a) haloperidol on food manipulation and (b) paroxetine on grooming. GAS-treated rats were impaired in manipulating food and haloperidol ameliorated this impairment, whereas paroxetine normalized the duration of induced-grooming. (a) Following a habituation session and a baseline session, rats underwent four additional food manipulation sessions separated by 3 wash-out days. Each rat in the haloperidol groups was injected with 0, 0.05, 0.1, or 0.15 mg/kg 1 h before testing. Each rat in the vehicle groups was injected with vehicle 1 h before testing on each of the 4 testing days. The figure presents the mean and SE of food manipulation scores of GAS (n=7) and control (n=8) rats treated with haloperidol, and of GAS (n=6) and control (n=6) rats treated with vehicle across all sessions. (b) The mean and SE of the duration of grooming during the three grooming sessions of GAS and control rats treated with 9.25 mg/kg paroxetine or vehicle 30 min before each session (n per group: control-vehicle=8, GAS-vehicle=8, control-paroxetine=6, GAS-paroxetine=10; note that the vehicle groups are the same as those appearing in Figure 1c). ^*Significantly different from the control-vehicle group, P<0.05; ^#Significantly different from GAS-drug group, P<0.005.

Figure 3

(a, b) IgG in GAS rat sera induced increased CaMK II activity in neuronal cells. CaMK II activation presented as percent of CaMK II activity above basal rate in (a) sera taken from GAS rats (n=8) and from control rats (n=10), (b) pooled rat serum (n=3) from GAS rats before absorption, following absorption with anti rat-IgG agarose beads and following absorption with BSA-agarose (also shown CaMK II activity with pooled control serum, n=3). ^*Significantly different from the control group, P<0.005; ^#Planned comparisons showed that GAS rat sera after the IgG absorption treatment were significantly different from pre-absorption GAS sera, P<0.005. (c, d) Dopamine D1 and D2 receptor membrane antigens were targeted by serum IgG antibodies in GAS-exposed rats. Sera IgG reacting with (c) dopamine D1 receptor and (d) dopamine D2 membrane receptor antigen (Perkin Elmer). Anti-D1/D2 dopamine receptor sera obtained commercially confirmed the dopamine receptor location on the blot as shown at the 50/51 kD molecular weight.

Figure 4

IgG deposition in brains of GAS-exposed rats. Thionin blue stained cell structures in transversal tissue sections taken through the striatum (a1, 4), cortex (b1, 4), thalamus (c1, 4), hippocampus (d1), and cerebellum (e1) of a GAS-exposed rat at a magnification of × 2.5 (1) and × 63 (4). IgG deposits in brain sections taken at the same level as (1) from a GAS (2, 5) and a control rat (3, 6) at a magnification of × 2.5 (2, 3) and of × 63 (5, 6). Tissue sections were incubated in biotinylated-anti-rat IgG and then incubated with avidin using Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Anti-IgG binding to brain sections was detected using diaminobenzidine for visualization of antibody deposition. Arrowheads illustrate the margins of regions of interest. Arrows mark staining of neuronal cells.

Figure 5

Anti-dopamine D1 and D2 receptor membrane antigen antibodies in SC and PANDAS. (a, c) Antibodies (IgG) in sera from SC (n=8) and PANDAS (n=27), but not attention deficit hyperactivity disorder (ADHD, n=5) or healthy controls (n=19), were significantly elevated against dopamine D1 (a) and D2 (c) receptor membrane antigens. Inset of a western blot in the graphs in (a) and (c) confirm IgG reactivity of PANDAS sera with purified human D1 and D2 dopamine receptor membrane antigen (50/51 kD) but not IgG in sera from healthy controls as shown as negative in the western blot. PANDAS sera used in the western blots had elevated antibody titers against D1/D2 membrane receptors (⩾8000) in the ELISA, whereas healthy controls had lower titers of antibody for D1 (500) or for D2 (2000) receptors. (b, d) Antibodies (IgG) in sera from three PANDAS patients were elevated against dopamine D1 (b) and D2 (d) receptor membrane antigens during active symptoms and were decreased during convalescence.