Subjects

All methods and procedures were approved by Institutional Animal Care and Use Committee (IACUC) of University of Medicine and Dentistry of New Jersey (UMDNJ), Newark, New Jersey. A total of 98 adult male Balb/c mice were used in this study. The mice were purchased from Charles River Laboratories (Wilmington, MA). We used male Balb/c mice, based on our findings in these mice that peripheral IL-6 administration induces behavior activating effects [18] and modulate brain dopamine and serotonin activity [20]. The mice were housed in standard polypropylene ‘shoebox’ cages in groups of four until testing, maintained on a 12 hour light/12 hour dark light cycle (7 am –7 pm), and permitted free access to standard laboratory chow and water.

Drugs

Recombinant human carrier-free soluble interleukin-6 receptor alpha (sIL-6Rα) (catalog #227-SR-025/CF) was purchased from R&D Systems (Minneapolis, MN). This form of sIL-6Rα is reactive to both human and mouse IL-6. Using human sIL-6Rα is advantageous as it permits the unambiguous identification of peripherally injected human sIL-6Rα in the mouse brain (Experiment 3). In these studies, anti-human IL-6Rα antibodies used to detect the human IL-6Rα did not detect the endogenous mouse IL-6R that may be present in the brain. The sIL-6Rα was injected subcutaneously (s.c.) (0–1 µg/mouse) in a volume of approximately 0.2 ml. The route of administration was based on our studies showing that monoclonal antibodies injected via the sc route gain entry into the brain and induce marked behavioral variations [23]–[27].

GBR 12909 (Tocris, Ballwin, MO), a highly selective dopamine transporter inhibitor with psychostimulant-like properties, was injected at a dose of 5 mg/kg (i.p.) in saline. We have determined that this dose induces a submaximal effect on motor activity and does not induce repetitive motor stereotypies; thus, one can determine whether combined treatment with sIL-6Rα and GBR 12909 has an additive effect on repetitive motor stereotypies.

Behavioral Testing

In Experiment 1, mice (n = 11/group) were injected with various doses of sIL-6Rα (0–1 µg/mouse, s.c.) and immediately thereafter were individually placed into a test arena (TruScan Behavioral Monitoring System; Coulbourn Instruments, PA) for 2-hr in normal illumination. A series of behavioral measurements were recorded including locomotion, vertical activity, horizontal and vertical stereotypic movements, jumps, and turns. Locomotion was defined as the sum of all vectored coordinate changes in the floor plane, and included floor plane total movement distance less the stereotypic movement distance. Turns were defined as movements where the animal enters 4 radially-contiguous quadrants in ascending or descending order, without interruption. Vertical stereotypic movements were defined as the total number of coordinate changes ±1.499 beam spaces in the vertical plane and back to the original point that do not exceed 2 seconds apart. Three such movements must be made before a stereotypy episode starts. When it does, the qualifying 3 movements are included in the total number of moves.

The test sessions were also filmed with a VHS camera, and at a later date, an experienced rater blinded to the treatment groups scored the incidence and duration of stereotypic behaviors, which included rearing, sniffing, head bobbing, and intense grooming. We selected these stereotypies based on our studies examining the behavioral consequences of cytokines and other immune-derived elements [18], [27], and the use of such behaviors as animal analogs of repetitive stereotyped movements [28]. Head bobbing was defined as the time (seconds) engaged in repeated movement of the head in the vertical head plane. A minimum of 3-seconds of uninterrupted head bobbing was defined as one episode. The duration of such episodes was recorded with a stopwatch. Measurements were taken 20-, 40-, 60-, 80-, 100- and 120- minutes after injection in 2-minute epochs. Results are presented as totals (± SEM) for the entire session, and in 20-min intervals (± SEM).

In Experiment 2, we determined whether sIL-6R treatment influences GBR 12909-stimulated stereotypy. Mice received saline or sIL-6Rα (0.5 µg/mouse, s.c.; n = 6/group; dose based on the findings of Experiment 1) and were individually placed into a holding cage for 30-min, whereupon they received an injection of a submaximal dose of GBR 12909 (5 mg/kg, i.p). The 30-min interval between injections was chosen to ensure that behavioral effects of sIL-6R and GBR 12909 would overlap. Immediately thereafter, the mice were individually placed into a test arena (TruScan Behavioral Monitoring System; Coulbourn Instruments, PA) for 2-hr as described earlier. Behavior was measured as per Experiment 1. We additionally rated behavior using a stereotypy rating scale [29].

Immunohistochemical analyses of peripherally administered sIL-6Rα in the brain

In Experiment 3, we determined whether peripherally injected human sIL-6Rα localize in brain regions associated with CSTC circuits, which are neuroanatomical substrates of the observed behavioral disturbances. Mice received single injections of human sIL-6Rα (0 or 0.5 µg/mouse, s.c.; n = 4/group) and immediately thereafter were individually placed into a test arena for 2 hours (as per Experiment 1). Immediately following the test session, the mice were deeply anesthetized with Na pentobarbital (60–80 mg/kg, IP) and transcardially perfused with 0.9% saline (pH 7.2) followed by 4% paraformaldehyde (pH 7.4). After perfusion, brains were removed from the skull and placed in 4% paraformaldehyde solution at 4°C for overnight. The brains were transferred to 30% sucrose solution and kept at 4°C until they sank to the bottom whereupon they were frozen in a cryostat (Leica CM1900) at −20°C for slow freezing. Sections were cut at 30 µm thickness and alternate sections were transferred to 24 well plates containing PBS and washed for 30 minutes. Brain sections were first blocked with 5% normal goat serum containing 0.3% Triton X-100 in PBS for 1 hour and then incubated with anti-human sIL-6Rα (Monoclonal Antibody) (1∶100 dilution) overnight at 4°C followed by incubation with goat anti-human IgG-FITC (green, 1∶200; Santa Cruz, CA) secondary antibody for 1 hour at room temperature. The slices were mounted on a slide, and viewed under an Olympus fluorescence microscope.

Statistics

The results were analyzed with both one-way and two-way (antibody receptor over time) analyses of variance (ANOVA) followed by Newman-Keuls test for confirmation of post-hoc comparisons (α = 0.05).

Effects of sIL-6R on novelty-induced motor activity

We determined whether a single peripheral injection of sIL-6Rα influence novelty-induced exploratory motor activity and repetitive stereotypies. sIL-6Rα administration induced significant increases in exploratory motor activity, including locomotion (F (3, 32)  = 3.76, p = 0.02; Fig. 1A), rearing (F (3, 29) = 3.74, p = 0.021) (Fig. 1C), and turning (F (3, 31)  = 6.42, p = 0.001; Fig. 1E). Newman-Keuls post-hoc comparisons (α = 0.05) revealed that compared to controls, the 0.5 µg dose of sIL-6Rα was most effective in inducing these effects. Of further importance, sIL-6Rα induced a pronounced increase in repetitive stereotypies, including head bobbing (F (3, 39)  = 3.80, p = 0.02; Fig. 2A), vertical stereotypy movements (F (3, 29)  = 3.38, p = 0.03; Fig. 2C) and vertical stereotypy episodes (F (3, 30)  = 4.32, p = 0.01; Fig. 2E).

Further examination of the data over 20-min intervals revealed time-dependent pattern of behavioral changes following sIL-6Rα injections. In particular, marked increases in novelty-induced exploratory motor behaviors were evident within 20–40 min of injection (Fig. 1B). Similar effects were observed for Rearing episodes (Fig. 1D) and Turning activity (Fig. 1F). While the magnitude of these effects declined thereafter, progressive increases in vertical stereotypies were expressed (i.e., 40–80 min post injection) and persisted for the remainder of the test session (Fig. 2B, 2D & 2F). The finding that the expression of repetitive stereotypies increased while exploratory motor behaviors declined implies selective effects on behavior.

Data recently published by our lab has shown that sIL-1R1 did not appreciably affect novelty-induced motor behaviors or repetitive stereotypies [30]. Thus, there is specificity with respect to the present behavioral effects induced by sIL-6R.

Effects of sIL-6R pre-treatment on GBR 12909-stimulated behavioral changes

In Experiment 1, we discovered that sIL-6R induce exploratory motor activity and repetitive stereotypies; this is consistent with an IL-6-like agonist effect. In Experiment 2, we determined whether sIL-6R pre-treatment would affect streotypies induced by the dopamine uptake inhibitor GBR 12909.

Compared to mice receiving saline + GBR 12909, mice receiving sIL-6Rα + GBR 12909 showed a significant increase in the combined stereotypy score (F (1, 14)  = 17.04, p = 0.001; Fig. 3). Inspection of the data over 20-min intervals revealed that effects persisted throughout the 2-hr test session Analyses of specific stereotypies revealed that mice in the sIL-6Rα + GBR 12909 group did not show the range of stereotypies induced by sIL-6Rα alone; thus, the present effects do not simply reflect a carryover effect of sIL-6R pre-treatment. Since GBR 12909 is a highly selective dopamine transporter inhibitor, these findings also imply a link between sIL-6R and brain dopaminergic system.

Localization of peripherally injected sIL-6Rs in brain regions associated with CSTC circuits

The findings of Experiments 1 and 2 imply that sIL-6Rα reaches brain regions associated with CSTC circuits, the putative neuroanatomical substrates of disorders associated with repetitive motor stereotypies. Previously, there was no evidence that peripherally injected soluble receptors localize in such regions. Accordingly, we determined whether peripherally injected human sIL-6R would localize in the cortex, striatum, and thalamus. As mentioned above, the anti-human IL-6Rα antibodies that were used to label IL-6Rα do not bind endogenous mouse IL-6R that may be present in the brain; thus, any labeled receptor represents exogenous sIL-6Rα.

In sIL-6Rα-treated mice, sIL-6Rα was found in the caudate-putamen (Fig. 4), nucleus accumbens (Fig. 5), motor and infralimbic cortices (Fig. 6 and 7), and ventral nuclei of the thalamus (Fig. 8). A striking finding was that sIL-6Rα co-localized with gp130 in these regions. Little or no label was detected in the ventral tegmental area, substantia nigra, or somatosensory cortex, although gp130 was expressed in these regions. These findings show a selectivity of sIL-6R labeling and perhaps suggest that sIL-6R does not target cell bodies within the mesolimbic and mesostriatal pathways.

Specificity of label is also demonstrated by the fact that human sIL-6R was not evident in saline controls. However, it is of interest that gp130 deposits were evident in saline-treated mice. Because saline controls were exposed to novelty stress for 2-hr immediately after injections, an intriguing possibility is that stressor exposure induced gp130 expression in the brain. To control for stress, we stained for gp130 in an additional group of controls that were not exposed to novelty stress. These control mice were deeply anesthetized immediately on arriving in the laboratory and transcardially perfused for subsequent IHC procedures. There was an absence of gp130 label in all of the brain regions examined in these unstressed controls (see Fig. 4, 5, 6, 7, and 8). These findings coupled with those in saline-treated mice exposed to novelty stress imply that stressor exposure induces gp130 expression in the brain.