Novel Object Recognition in Rats With NMDAR Dysfunction in CA1 After Stereotactic Injection of Anti-NMDAR Encephalitis Cerebrospinal Fluid

Abstract

Purpose: Limbic encephalitis associated with autoantibodies against N-methyl D-aspartate receptors (NMDARs) often presents with memory impairment. NMDARs are key targets for memory acquisition and retrieval, and have been mechanistically linked to its underlying process, synaptic plasticity. Clinically, memory deficits are largely compatible with a pre-dominantly hippocampus-dependent phenotype, which, in rodents, is principally involved in spatial memory. Previous studies confirmed the impaired spatial memory in the rat model of anti-NMDAR encephalitis. Here, we hypothesized that non-spatial memory functions, such as object recognition might also be affected in this model. Methods: We performed stereotactic intrahippocampal bolus injection of human cerebrospinal fluid (CSF) from anti-NMDAR encephalitis and control patients into the hippocampus of the anesthetized rat. After recovery for 1-8 days, hippocampal slices were prepared from these animals and NMDAR-dependent long-term potentiation was assessed at the Schaffer collateral-CA1 synapse. In addition, we performed behavioral analyses using the open field and novel object recognition tasks. Results: NMDAR-dependent long-term potentiation in the hippocampal CA1 area was significantly suppressed, indicating successful NMDAR dysfunction in this subfield. Spontaneous locomotor activity as well as anxiety-related behavior in the open field did not differ between NMDAR-CSF-treated and control animals. In the novel object recognition task, there were no differences in the motivation to approach objects. In contrast, we observed a significantly preferred exploration of the novel object only in control, but not in NMDAR-CSF-treated rats. Conclusion: These results indicate that NMDAR dysfunction obtained by intrahippocampal stereotactic injection does not alter locomotor or anxiety-related behavior. In addition, approach to an object or exploratory behavior in general are not affected either, but intact initial NMDAR-dependent processes might be involved in novel object recognition.

Keywords: anti-NMDAR encephalitis; cerebrospinal fluid; long-term potentiation; object recognition; perirhinal cortex.

Figure 1

LTP deficit in hippocampal CA1. (A) Immunofluorescence micrographs showing the marker dispersion in the hippocampus 1 h after injection into CA3 stratum radiatum (denoted by an arrowhead), magnification 20×. Note that the marker intensely diffuses into the dentate gyrus, but also reaches CA1 and the parahippocampal gyrus. The white boxes indicate the positions of enlarged micrographs (magnification 200×): CA1, Cornu Ammonis 1; MEC, medial entorhinal cortex; LEC, lateral entorhinal cortex; PER, perirhinal cortex. The scale bar indicates 1,000 μm. (B) Schaffer collateral–CA1 synaptic long-term potentiation (LTP) is significantly reduced in slices from NMDAR-CSF-treated rats. Representative traces (B₁) were taken at the time-points indicated in the time course (B₂). The arrow indicates the time-point of delta-burst stimulation (dBS). (C) There was no significant correlation between the LTP level and the post-operative day in both groups. (D) Box plot showing the LTP magnitude of all groups (NMDAR-CSF: N1–3; control-CSF: C1–3; naive) at the end of the experiment (**P < 0.01). Experiments in the presence of the NMDAR blocker D-AP5 (indicated by “A”) are presented with dotted lines.

Figure 2

Open field and novel object recognition. (A) Time frame of the behavioral experiment. The open field test was performed on post-operative day 4 or 5 (here only shown for animals which started on post-op day 4). Object recognition was tested on three consecutive days. Note that sample phase 2 and choice test 1 are on the same day with a delay of 1.5–3 h. (B) Scheme of the arena (100 × 100 cm) with trajectory of a control animal (group C5) inserted within the center which stays at the 20 cm rim for almost the complete period. (C) Time spent at the rim (20 cm) of the arena was not different between all three groups. (D) Total distance covered during 5 min was also not different between all three groups. (E) Scheme of the arena equipped with two objects, and exploration was defined as presence of the animal close to the object (diameter 18 cm, gray zone). (F) Time spent with the objects in NMDAR-CSF-treated and control rats during the behavioral experiment. (G) NOR index for choice test 1. Note that random presence of the animal at the objects would lead to a NOR index of 50%. The asterisk indicates a significant difference of the observed values against this chance level of 50%. Two animals (one NMDAR-CSF-treated and one control rat) spent no time at both objects during the choice test, these animals were not included for the NOR index calculation (i.e., n = 13 instead of 14). (*P < 0.05).