Effects of sustained i.c.v. infusion of lupus CSF and autoantibodies on behavioral phenotype and neuronal calcium signaling

Abstract

Systemic lupus erythematosus (SLE) is a potentially fatal autoimmune disease that is often accompanied by brain atrophy and diverse neuropsychiatric manifestations of unknown origin. More recently, it was observed that cerebrospinal fluid (CSF) from patients and lupus-prone mice can be neurotoxic and that acute administration of specific brain-reactive autoantibodies (BRAs) can induce deficits in isolated behavioral tasks. Given the chronic and complex nature of CNS SLE, the current study examines broad behavioral performance and neuronal Ca²⁺ signaling in mice receiving a sustained infusion of cerebrospinal fluid (CSF) from CNS SLE patients and putative BRAs (anti-NR2A, anti-ribosomal P, and anti-α-tubulin). A 2-week intracerebroventricular (i.c.v.) infusion of CSF altered home-cage behavior and induced olfactory dysfunction, excessive immobility in the forced swim test, and perseveration in a learning task. Conversely, sustained administration of purified BRAs produced relatively mild, both inhibitory and stimulatory effects on olfaction, spatial learning/memory, and home-cage behavior. In vitro studies revealed that administration of some CSF samples induces a rapid influx of extracellular Ca²⁺ into murine neurons, an effect that could be partially mimicked with the commercial anti-NR2A antibody and blocked with selective N-methyl-D-aspartate (NMDA) receptor antagonists. The current findings confirm that the CSF from CNS SLE patients can be neuroactive and support the hypothesis that intrathecal BRAs induce synergistically diverse effects on all domains of behavior. In addition, anti-NMDA receptor antibodies may alter Ca²⁺ homeostasis of central neurons, thus accounting for excitotoxicity and contributing to the heterogeneity of psychiatric manifestations in CNS SLE and other autoantibody-related brain disorders.

Keywords: Anti-NMDA receptor; Anti-ribosomal P; Anti-α-tubulin; Autoimmunity; Brain-reactive autoantibodies; CNS SLE; Cerebrospinal fluid.

Fig. 1

Technical details of survival surgery. a All mice underwent unilateral implantation of a sterilized cannula into the right lateral ventricle and subcutaneous implantation of a primed Alzet mini-pump, connected to a cannula via vinyl catheter tubing. Both groups received artificial CSF (aCSF) for 4 days to facilitate postoperative recovery. Hereafter, infusion of the solution of interest was initiated [CNS SLE or control CSF samples in Study 1, purified brain-reactive autoantibodies (BRA) or aCSF in Study 2] and continued for 2 weeks. An oil drop “spacer” was used to prevent mixing of aCSF in the tubing and the experimental solution in the primed pump. b An animal moving freely following survival surgery. c Histological verification of coordinates obtained by post-mortem injection of Toluidine blue into the vinyl tubing cut at the neck level. d Verification of antibody diffusion: control section of the dry ice-fixed contralateral periventricular region after the 2-week i.c.v. administration of CNS SLE serum (e) and the same region in another brain showing diffusion gradient in fluorescence when CNS SLE serum was premixed with DyLight 488. Note: Images were digitized using an Axioskop 2 Plus microscope with a 5× objective and AxioVision 4.6 software (Carl Zeiss, Inc., CA, USA)

Fig. 2

Schematic representation of the experimental design. Prior to testing, all mice were tail-tattooed and habituated to the experimenters. After being assigned to two behaviorally comparable groups, they underwent survival surgery and an identical sequence of tests. The behavioral battery was designed to evolve from less towards more strenuous tasks to mitigate residual stress effects on subsequent tests. Abbreviations: T – Tattooing; H – Habituation; INBEST – Integrated Behavioral Station; SAB – Spontaneous Alternation Behavior; SDT – Step-Down Test; NO – Novel Object Test; FST – Forced Swim Test; OF – Open Field Test; MWM – Morris Water Maze; OS – Olfactory Sensitivity; OM – Olfactory Memory; OD – Olfactory Discrimination; BW – Beam-Walking test; RR – Rotarod

Fig. 3

Post-surgery effects of CSF administration on body weight and home-cage behavior in the Integrated Behavioral Station, INBEST. a Treatment with lupus CSF was accompanied by a transient, but significant reduction in body weight when CSF infusion began 4 days after pump implantation. In comparison to control mice treated with NMO CSF, experimental animals exposed to CNS SLE CSF (b) drank less water and (c) consumed less food in INBEST during the 10-h testing period (Days 4 – 18). They also showed (d) lower running wheel activity levels, (e) prolonged stay in the shelter, and (f) decreased ambulation in the home-cage environment (n = 8 mice/group)

Fig. 4

Post-surgical effects of CSF infusion on olfactory function, forced swimming and performance in learning/memory tests. a When initially exposed to the olfactory discrimination paradigm during baseline assessment, mice assigned to receive CNS SLE CSF spent significantly more time investigating cinnamon in their first trial but performed comparably to control animals in subsequent exposures. b Following surgery, both groups seemed to habituate to repeated exposures to the cinnamon-scented filter paper. However, CNS SLE CSF-treated mice spent significantly less time investigation paprika-laced filter paper in the final (dishabituation) trial. c When forced to swim in an empty pool for 10 min, the sustained administration of CSF from CNS SLE patients reduced overall swimming distance and d increased floating. e Control animals receiving NMO CSF exhibited a significant post-surgery drop in spontaneous alternation rates in the T-maze that was not noted in animals treated with CNS SLE CSF. f Despite similar path lengths to locate the cued platform on Day 1, control animals swam longer distances to find a submerged platform on subsequent acquisition trials in the Morris water maze. g Even though NMO CSF-treated animals showed relative deficits in acquiring the location of a hidden platform, administration of CNS SLE CSF induced increased perseveration when the platform was re-located and submerged in reversal trials (n = 8 mice/group). Abbreviations: Cin – Cinnamon; Pap – Paprika; Acq – Acquisition; RevCue – Reversal Cue; RevAcq – Reversal Acquisition

Fig. 5

Behavioral effects of anti-NR2A antibody administration. a This treatment resulted in a significant reduction in time spent exploring cinnamon and paprika in the olfactory discrimination task. These results suggest that anti-NMDA receptor binding attenuates responsiveness to previously presented scents. b Spatial learning/memory assessment in MWM revealed similar performance outcomes in cued and hidden versions of the test. However, when the platform was relocated to the opposite quadrant, mice administered anti-NR2A antibodies exhibited significantly shorter path lengths to locate it. c They also swam shorter distances in an empty pool devoid of a platform during probe and extinction trials (n = 12 mice/group). Abbreviations: Cin – Cinnamon; Pap – Paprika; Acq – Acquisition; RevCue – Reversal Cue; RevAcq – Reversal Acquisition; Ext – Extinction

Fig. 6

Behavioral effects of sustained anti-RPLP0 antibody administration. a Administration of anti-RPLP0 antibodies into the brains of healthy mice led to a transient decrease in water consumption that was prominent on the last day of INBEST testing. b These mice also displayed a transient, but significant reduction in preference for 8% sucrose solution, as compared to their baseline performance. c When exposed to the MWM, no group differences were detected in the latency to locate a cued or hidden platform in acquisition and reversal trials. d However, ARPA-treated mice displayed poorer recall when the platform was removed from the pool during the probe trial (n = 12 mice/group). Note: Eight animals had to be excluded from the analysis of the probe and extinction trials due to a technical error with video-tracking software. Abbreviations: INBEST – Integrated Behavioral Station; Acq – Acquisition; RevCue – Reversal Cue; RevAcq – Reversal Acquisition

Fig. 7

Behavioral effects of sustained anti-α-tubulin antibody administration. a Prolonged infusion of anti-α-tubulin antibodies increased post-surgical home-cage activity, as measured by increased number of running wheel rotations, b more time spent in the running wheel, and c less time spent in the shelter. d In the MWM, mice had similar latencies to locate cued and hidden platforms. However, relocation of the platform in reversal acquisition trials resulted in significantly shorter escape latencies in mice treated with anti-cytoskeletal antibodies. Together with home-cage behavior, these results suggest that sustained administration of anti-α-tubulin antibodies has a stimulatory effect on certain behaviors (n = 12 mice/group). Abbreviations: INBEST – Integrated Behavioral Station; Acq – Acquisition; RevCue – Reversal Cue; RevAcq – Reversal Acquisition

Fig. 8

Examples of CSF-induced intracellular calcium transients in cultured hippocampal neurons. a Brightfield (transmitted light) image of cultured hippocampal neurons. b Fluorescent image of hippocampal neurons loaded with 5 μM Fluo-4 AM, in color-coded intensity scale (0–255, right) before the application of CSF. c An example of intracellular calcium peak response to CNS SLE CSF #4, 3 s after application. Scale bars for (a–c) are shown in lower right corner and cell bodies of neurons and astrocytes are indicated by arrows and asterisks, respectively. d–j Representative traces of intracellular calcium responses (normalized fluorescence intensity) of hippocampal neurons challenged by CSF from CNS SLE or NMO patients. CSF application is indicated by a black dot below the trace. CSF origin, dilution, as well as the presence/absence of external calcium is indicated above the traces. In an external solution with 2 mM Ca²⁺, 1:25 CNS SLE CSF #4 induced two types of responses, with (d) only fast or with (e) fast and late slow components. f The same sample at 1:50 dilution was also able to increase the cytosolic calcium concentration in the repeated application, to the same level as the first response. g 1:10 CNS SLE CSF #2 induced a fast calcium transient in 2 mM Ca²⁺. h Disease control NMO CSF in 1:10 dilution did not influence the intracellular calcium concentration in 2 mM Ca²⁺ external solution. i In a Ca²⁺-free external solution, 1:25 CNS SLE CSF #4 did not induce the fast transient, and j no calcium response could be detected after application of 1:10 CNS SLE CSF #2. Note: Calibration for (d–j) is shown in the lower right corner

Fig. 9

Effects of blockers of voltage-gated calcium channels and inhibitors of ionotropic glutamate receptors on CNS SLE CSF #4 - induced calcium transients. a A representative trace of normalized fluorescence with gradually decreasing amplitudes following differential drug treatments to block action potentials (TTX), voltage-gated calcium channels (VGCC_block) and AMPA/kainate and NMDA receptors (GluR_inh). b Summary histogram of peak amplitudes confirming that blockade of voltage-gated and ionotropic receptors decreases the amplitude of the control response. c Representative trace and d summary histogram of CNS SLE CSF #4 - induced calcium transients showing that GluR_inh drugs alone lower the amplitude of the control response. e NMDA receptor blockade with 200 μM DL-AP5 completely abolished the control response, suggesting a predominant role for NMDA receptors in CNS SLE CSF-induced calcium transients. The addition of CSF in (a, c, e) is indicated by black dots below the trace. Drugs used are indicated by the gray timelines above the trace and calibration within the trace on the right. * p < .001, two-tailed paired t-test. Abbreviations: TTX – Tetrodotoxin; VGCC_block – GVIA + AGA + NIF; GVIA – ω-conotoxin GVIA; AGA – ω-agatoxin TK; NIF – Nifedipine; GluR_inh – CNQX + D-AP5; CNQX – 6-Cyano-7-nitroquinoxaline-2,3-dione; D-AP5: D-2-Amino-5-phosphonopentanoic acid; DL-AP5 – DL-2-Amino-5-phosphonopentanoic acid

Fig. 10

Comparison of averaged intracellular calcium transients evoked by effective CNS SLE CSF and commercially available antibodies in time. a Trace illustrating that CNS SLE CSF #2 diluted to 1:10 evoked only a fast transient that peaked 3 s after CSF application. b The calcium response to 1:25 CNS SLE CSF #4 exhibited a fast transient after 3 s that was followed by a slower component measured 3 min after application and a return to basal levels after wash. c Anti-NR2A evoked a fast calcium transient that peaked at 6 s and a slow component that peaked after 3 min and could not be washed. d Anti-RPLP0 failed to induce a fast transient but provoked a slow and sustained rise of [Ca²⁺]_i 3 min after application. The application of CSF/antibodies in (a–d) is indicated by an upward arrow, duration by the gray line above the trace, wash by a thick downward arrow, and depolarizing 5 s pulse (50 mM K⁺) by a black strip above the trace. The origin and dilution of CSF samples, as well as the antibodies and concentration used, are indicated above the gray line