Persistent microglial activation and synaptic loss with behavioral abnormalities in mouse offspring exposed to CASPR2-antibodies in utero

Plasma exchange samples (obtained during periods of disease exacerbation) of two male patients with CASPR2-positive encephalitis (CASPR2-IgG) and sera kindly donated from three healthy age- and sex-matched individuals (HC-IgG) were used in this animal model. Written informed consent was obtained from both patients and healthy controls. Human IgG was purified through the ammonium sulphate precipitation method to avoid possible damage to the Fc domains during elution from Protein-G columns [17]. The preparations were extensively dialyzed against Hartmann’s solution, concentrated by dialysis against polyethylene glycol, and filter-sterilized. IgG concentration was determined using a commercially available human IgG radial immunodiffusion kit (The Binding Site, Birmingham, UK). Antibody levels were determined using a live cell-based assay expressing EGFP-tagged human CASPR2, as previously reported [13]. A summary of the details of the patients, plasma antibody titers, number of injected dams, and injected material for each experiment are provided in Online Resource 1 and 2.

Specificity of binding to CASPR2 was assessed for all samples used. The two CASPR2-antibody patients’ IgGs (CASPR2-IgGs) were shown to bind strongly to the surface of live cells expressing EGFP-tagged human CASPR2, to live (unfixed) rodent hippocampal neurons, and to sections of fixed mouse brain from wild-type mice. There was no binding to live hippocampal neurons or to brain sections from Cntnap2 knockout mice (B6.129(Cg)-Cntnap2^tm1Pele/J; Jackson Laboratory), a homozygous knockout mouse for the gene encoding CASPR2, confirming that there was absent or limited reactivity with other neuronal targets in the CASPR2-IgG preparations (Fig. 1a, top panels). Healthy control-IgG 1-3 did not bind to human CASPR2-EGFP-transfected cells, mouse live hippocampal neurons, or brain tissue sections (Fig. 1a, bottom panels). Plasma CASPR2-antibodies from both patients bound to wild-type fetal brain tissue at E18.5 (Fig. 1b). In the isocortex, human IgG from CASPR2-antibody patients was detected around the neuronal cell body and dendritic processes, extending radially to the deeper layers, as already reported for CASPR2 expression [11]. This staining was absent in the Cntnap2 knockout mice when CASPR2 or HC plasma was used.

Crl: MF1 outbred female mice (20–25 g) and adult male breeders of the same strain were purchased from a licensed breeding establishment (Charles River, Kent, UK Ltd). The animals were housed and mated in the Biomedical Services Unit at the John Radcliffe Hospital under standard laboratory conditions (ad libitum access to food and water; 12:12 light:dark cycle with lights on at 7 am). Establishment of pregnancy relied on detection of vaginal coagulant plugs the morning after mating. E0.5 was defined as the day on which the plug was detected and P0 as day of delivery. All in vivo experiments, with IgG preparations coded and investigators blinded, were performed in accordance with the United Kingdom Home Office Animals in Scientific Procedures Act (1986) and in accordance with institutional guidelines under HO PPL 40/3581.

There were three main experiments (Experiment 1–3). In each, dams were tagged and randomized between cages, and injected intraperitoneally with independently coded purified human IgG preparations (15–20 mg/day; volume 1–2 ml) daily from E12.5 until E18.5 or until the time of culling [15], as indicated in Fig. 2a.

Experiment 1 assessed the maternal-to-fetal transfer of antibodies. Fifteen milligrams of one CASPR2-IgG was injected daily into four pregnant dams from E12.5 onwards. Dams were sacrificed at E14.5, E16.5, E18.5, and P0 (one dam per time point) by CO₂ anaesthesia, followed by cardiac puncture for blood collection. The dams were then dissected and the uteri removed. Fetuses were dissected out of their membranes, separated from the placenta, and washed in saline. Fetuses/pups were sacrificed by decapitation, and fetal blood was collected by suspending the trunk. Blood was pooled from the fetuses/pups of each litter and the sera separated and stored at −20 °C. Total human IgG was determined by quantitative western blotting and CASPR2-antibodies detected by a live cell-based assay as above (details in Online Resource 3).

Experiment 2 examined the embryos at E18.5 after injection of three dams per group with 20 mg IgG per day. Dissected fetuses were decapitated and flash frozen in ice-cold isopentane. Twelve micrometer sagittal sections of the entire head were obtained from 3 fetuses per treatment group, from 3 different litters, and used for immunohistochemistry (IHC)/immunofluorescence. Details are provided in Online Resource 3.

Brain sections (1 in 10 series) from one embryo per litter were stained for total human IgG. In the same slide containing 4 consecutive sections, the two bottom sections were briefly washed 3 times in phosphate buffer saline (PBS), while the two upper sections were unwashed. All sections were then fixed with 4% paraformaldehyde (PFA) for 10 min. After 3 washes in PBS, sections were incubated with CF488A anti-human IgG (Biotium, 20022) at 1:500 overnight at 4 °C. The following day, sections were washed 3 times in PBS and incubated with rabbit anti-von Willebrand factor (VWF; Millipore, AB7356, 1:250) for 3 h at room temperature (RT), followed by incubation with goat anti-rabbit Alexa Fluor 568 secondary for 1 h at RT. After washing 3 times in PBS, coverslips were mounted using fluorescent mounting media containing DAPI (1:1000). Sections were visualized using a Leica DM 2500 immunofluorescence microscope. For quantitative analysis of the mean fluorescent intensity, 3 photomicrographs were taken from the isocortex in 3 different sections from 3 embryos/treatment group (27 photomicrographs/treatment group). Mean fluorescence intensity was then assessed for each photomicrograph using ImageJ. Results were plotted for both individual photomicrographs and for the mean results from each pup.

Human IgG was eluted from CASPR2-IgG and HC-IgG exposed embryos (3 embryos per group) following a previously described protocol [6] and as detailed in Online Resource 3.

Experiment 3 looked at the long-term effects of maternal–fetal transfer. Dams were injected daily from E12.5 to E18.5 with CASPR2-IgG from two patients and HC-IgG from two donors (4 dams per treatment group; IgG concentration 20 mg/day; volume 1–2 mL/day) and allowed to deliver. At P0 (usually the morning following delivery), the number of live and dead pups was counted and the litter left undisturbed with the dam for 24 h. At P1, to ensure equal maternal interactions, six pups were randomly selected from each litter and individually tattooed for coded behavioral testing. During these procedures, the dam was left in the home cage and was placed in the testing room for habituation. The remaining pups from that litter were culled by decapitation and blood was pooled for antibody testing.

Behavioral experiments were conducted at the times indicated in Fig. 2b and c. Behavioral testing of the neonatal pups was performed from postnatal days 1–21 and consisted of evaluation of developmental milestones using the modified Fox battery [12], which assesses physical maturation readouts, neurological reflexes, and motor coordination, and a maternal retrieval test, as described in Online Resource 3.

A battery of behavioral tests was performed in the mice when adults (>4 months old, mainly 4–8 months). Tests performed assessed locomotor activity, motor coordination, anxiety, short-term memory, social interaction/activities and non-social behaviors, and olfaction (see Online Resource 3). All behavioral tests and subsequent histological analyses were performed blinded to treatment group.

After behavioral testing, six animals per treatment group (3 males and 3 females) were randomly selected for histological analysis at 12 months. Mice were deeply anaesthetized with Isoflurane followed by pentobarbital injection (100 mg kg⁻¹ ip) and transcardially perfused with 100 ml of PBS followed by 50 ml of ice-cold 4% PFA in 0.1 M phosphate buffer. Brains were removed and post-fixed for 24 h at RT before transferring to 30% sucrose in PBS. Using a freezing stage sledge microtome, free-floating coronal sections were obtained in 15 series at a thickness of 50 μm/section.

The detailed methods for cortical layer measurements and immunohistochemistry for neurons and microglia are given in Online Resource 3. CUX-1-positive cells in layers V and VI of the somatosensory cortex were counted in the coronal slices at Bregma—1.46 mm. 16 images (418.4 μm × 418.4 μm) were taken per mouse using a Zeiss confocal microscope in one plane (depth = 5 μm) on 50 μm coronal sections incubated in fluorescently tagged antibodies. At least 350 cells were counted per brain using Fiji cell counter and an average density (cells/mm²) per case was obtained.

Quantification of activated microglia (defined as CD68/Iba-1 positive cells) was performed in layers I, II–III, and V–VI in the infralimbic and prelimbic areas (Bregma +1.42 mm) and in layers I, II–IV, and V–VI in the somatosensory cortex (Bregma −1.46 mm). The z-stack area was 174.3 μm × 174.3 μm for the prefrontal areas and 111.15 μm × 111.15 μm for the somatosensory areas. The z-step/interval was 2 µm and microglial cells were counted within a 50 µm depth. Four z-stacks per layer were imaged in the infralimbic and prelimbic areas and six z-stacks per layer in the somatosensory area. An average density was obtained (cells/mm³) in both hemispheres. At least 100 cells per layer were counted.

Microglial morphology was assessed in 24 confocal z-stacks/case at 40× magnification (28 μm thickness with imaging every 2 μm) detecting fluorescence in IBA-1 expressing cells in layers of the somatosensory cortex. Minimum resolution of 512 × 512 was used and with a line averaging of 4 to allow for a detailed assessment of microglial cells in each group. A macro (details in Online Resource 3) written for use in Fiji (Image Analysis software) was used to obtain in each stack a clear representation of the cell body and processes of 101 microglial cells per group. Soma size (μm²) and number of ramifications per cell were recorded manually in Fiji.

Immunohistochemistry for synaptic proteins was performed using a guinea pig anti-synaptophysin antibody (Frontiers Institute, Syn-GP-Af300; 1:200) and a rabbit anti-PSD95 antibody (Frontiers Institute, PSD95-Rb-Af628; 1:200) to identify the presynaptic zone and the post-synaptic density of asymmetric glutamatergic synapses, respectively. For GluA1/PSD95 immunofluorescence, a rabbit anti-GluA1 antibody (Frontiers Institute, GluA1-Rb-Af692; 1:200) and a guinea pig anti-PSD95 antibody (Frontiers Institute PSD95-GP-Af660; 1:200) were used. 50 μm sections were air-dried for 48 h, rehydrated in PBS for 15 min, and then placed in a solution of 50% ethanol: PBS for 30 min at RT. Sections were subsequently dipped in double distilled H₂0 to wash off the ethanol then in citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween-20 at pH 6.2) prior to a heat-mediated antigen retrieval step in citrate buffer for 10 min. Proteinase K treatment (4 μg/ml) for 10 min followed by a 10 min pepsin treatment (1 mg/ml) at 37 °C in 0.2 M HCl solution were required to unmask antigenic epitopes. Sections were then incubated with primary antibodies diluted in PBS-Azide-Triton-X-100 (0.3%) for 3 days at RT, and washed and incubated overnight with anti-guinea pig (1:500) and anti-rabbit (1:500) secondary antibodies from Life Technologies conjugated to Alexa Fluor 555 and 488 fluorophores, respectively, and concentrated DAPI at 1:50,000 in PBS. Sections were subsequently washed and mounting medium added. Two z-stacks measuring 66.11 × 66.19 × 6 μm with a voxel size of 0.04 × 0.04 × 0.1 μm (XYZ) were taken in each layer of the infralimbic, the prelimbic, and the somatosensory cortices in both hemispheres. This resulted in 60 optical sections available to quantify the number of PSD95-positive profiles and GluA1-positive profiles (synaptic contained in PSD95-positive profiles and extra-synaptic negative for PSD95) and at least 2000 puncta per layer were quantified. All image processing and analyses was performed in Fiji. To process images for quantification of synaptic profiles, first z-stacks were deconvolved using the WPL deconvolution algorithm in the Parallel Iterative Deconvolution ImageJ plugin [38] based on the Iterative Deconvolve 3D algorithm [27] utilizing a theoretical 3D point spread function generated in PSF Lab [19]. Image stacks were then filtered with a 3 × 3 median filter and thresholded using the OTSU method [20] to obtain accurate binary representations of positive profiles. To assess synapse profiles, individual z-slices separated by 0.4 μm were isolated and positive puncta profiles were analyzed using the Analyze Particles algorithm in Fiji. Puncta profile counts in each layer were averaged over two sections per animal, and plotted as profile counts per 100 μm².

NeuN-positive cells were quantified by layer in the coronal slice at Bregma—1.46 mm. The z-stack area was 174.3 μm × 174.3 μm for the medial prefrontal areas and 111.15 μm × 111.15 μm for the somatosensory areas. The z-step/interval was 5 μm and NeuN-positive cells were counted within eight optical sections through the 50-μm stack. Four z-stacks per layer were imaged in the infralimbic, prelimbic, and somatosensory areas. An average density was obtained (cells/mm³) in both hemispheres. For layer I, at least 100 cells were counted; for the remaining layers, at least 400 cells/layer were counted.

Results were analyzed by independent samples t test or a Mann–Whitney U test, depending on data distribution, or by repeated-measures/two-way ANOVA. A Holm–Sidak correction was applied to correct for multiple comparisons when appropriate. A statistically significant result was considered if p < 0.05. Analysis was performed in IBM SPSS statistics version 21.0 (SPSS Inc., Chicago, USA). Graphs were plotted using Graph Pad prism version 6 (GraphPad software, San Diego, California, USA). To account for intra-litter effects during the neonatal period, the means for each litter were used in statistical comparisons, rather than the data from individual pups. In adult animals, unless mentioned otherwise, data represent the mean ± SEM of average values derived from individual animals.

The maternal human total IgG levels varied between 3 and 7 mg/ml over the 7 days of transfer (E12.5–E18.5), while human IgG in the fetal sera did not exceed 0.4 mg/ml at the end of gestation (i.e., about 10% of maternal levels at that time; Fig. 3a, left panel). Nevertheless, the CASPR2-antibodies were clearly present in the pool of fetal sera, with titers at the end of gestation of 1:40 in Experiment 1 (Fig. 3a, left panel) and 1:100–1:200 in the pups in Experiments 2 and 3 (data not shown), where higher titer IgG preparations were used.

To see whether human IgG could be detected in the brain tissue of the fetal brains shortly before delivery (Exp 2), three dams per treatment group were injected with HC-IgG or CASPR2-IgG (patient 1, 20 mgs/day) from E12.5 to E17.5, and the dams culled at E18.5. Visual inspection of all embryos prior to culling showed no gross malformations in the litters of either treatment group, and there were no gross brain morphological abnormalities on Nissl staining of the embryo heads after snap-freezing (Online Resource 4).

When the fetal brain sections were PFA-fixed on the slide, intense deposition of human IgG was observed in the fetal vessels [co-labeled with anti-von Willebrand factor (VWF) antibody], choroid plexus, and the periventricular zones in both experimental groups (Fig. 3b, panel i), as expected from circulation of the IgG. However, there was greater deposition of human IgG in the perivascular areas of the CASPR2-IgG than in the HC-IgG exposed fetuses suggesting that some might be bound in situ (Fig. 3b, panel ii). This was confirmed when adjacent sections were washed gently to remove unbound IgG before fixation; in CASPR2-IgG—but not HC-IgG exposed brains—human IgG was clearly bound in the parenchyma (Fig. 3b, panel iii). The lack of detectable bound IgG in three saline-exposed offspring, on either fixed or unfixed tissue, confirmed the specificity of the binding for human IgG (Fig. 3b, i–iii).

These observations were quantified by measuring the mean fluorescence intensity in the isocortex. Despite some variability between sections of the same embryo, the fluorescence intensity in the CASPR2-IgG exposed embryos was higher than in the HC-IgG exposed embryos, considering either the mean fluorescence intensity of all photomicrographs [t(33.2) = 4.2, P < 0.0001] or the mean for each embryo [t(4) = 4.54, P = 0.01; Fig. 3b, panel iv]. Thus, IgG remained bound to the brain tissue in the CASPR2-IgG exposed. To establish whether this bound IgG was directed against CASPR2, acid elution of the bound human IgG was performed (n = 3 animals/group), following a previously described method [6] (Fig. 3c). CASPR2-antibodies were detected in the eluate from CASPR2-IgG but not from HC-IgG exposed embryos. However, at this gestational age, a reduction in CASPR2 expression was not detected in the CASPR2-IgG exposed embryos in comparison with HC-IgG exposed embryos by western blotting (Online Resource 5).

For behavioral testing, four dams per treatment group were injected daily (two with patient 1 and two with patient 2) from E12.5 to E18.5 and allowed to deliver. HC-IgG and CASPR2-IgG exposed litters showed no differences in the number of surviving pups (U = 6; Z = −0.59; P = 0.69) and no obvious gross abnormalities. To ensure that the behavioral results were not influenced by different litter sizes, which could affect maternal–offspring interactions, each litter was randomly culled to 6 mice at P1. Gender of the randomly selected pups for study did not differ between groups (Fisher’s exact test P = 0.39; Fig. 4a).

There was no difference in weight increase over the postnatal period (group × time F(2.8, 127.2) = 0.27, P = 0.51, Greenhouse–Geisser correction; group F(1,46) = 0.28, P = 0.6; Fig. 4b). The maternal retrieval test, which assesses maternal-infant interactions, showed variation but no differences between groups in the time taken by the dam to return all pups to the nest [t(6) = 1.2, P = 0.46], or in the latency to retrieve the first pup [t(6) = 0.8, P = 0.29; Fig. 4c]. There were no differences in any of the parameters analyzed by the modified Fox battery, including physical maturation readouts, neurodevelopmental reflexes, or the motor coordination performance (Fig. 4d); a modest increase in days to reach rooting behavior was not significant.

The mice from Experiment 3 were maintained in their cages and studied between ages 4–10 months. There were no significant differences in tests of locomotor activity (Fig. 5a), motor coordination (accelerating rotarod; Fig. 5b) or in anxiety tests (elevated plus-maze and light–dark box; Fig. 5c/d), although a trend towards less time spent in the light was noted in the CASPR2- IgG exposed mice (P = 0.07; Fig. 5c/d).

Social behavior was assessed using two methods at 6–8 months. In the three-chamber social interaction test, both CASPR2-IgG and HC-IgG exposed mice showed a preference to spend time exploring the wire cage containing a novel, unfamiliar mouse compared with an empty wire cage, but CASPR2-IgG exposed mice spent significantly less time interacting with the mouse than HC-IgG exposed mice [t(22) = 3.086, P = 0.006; raw data available in Online Resource 6]. When subsequently given a choice between the same mouse and a new mouse, both CASPR2-IgG and HC-IgG exposed mice preferentially explored the novel animal suggesting no social memory deficits [t(22) = 1.253, P = 0.223; Fig. 5e]. Using the reciprocal social interaction test, CASPR2-IgG exposed mice were again found to spend less time engaged in active social interaction (sniffing, licking, mounting, or crawling under or over the other mouse; Fig. 5f) than the HC-IgG exposed mice (U = 11, Z = −2.647, P = 0.008), confirming a significant deficit in social interaction in those mice exposed to CASPR2-IgG in utero.

Importantly, there were no differences between groups in measures of locomotor activity during the period of habituation to the testing arena, ensuring that the decrease in social interaction was not due to differences in exploratory behavior or habituation to the testing environment (Online Resource 7). Furthermore, there were no differences in an olfactory test, with both groups showing an equivalent preference for a novel odor compared to a recently experienced, and thus familiar, odor (Fig. 5g). In addition, there were no differences between groups in the time spent exploring the odor area during the sample phase of this test [t(14) = 0.55, P = 0.6; data not shown]; these results confirmed that the social deficits observed were not due to reduced olfaction or response to novelty.

Non-social behaviors (grooming, digging) were also assessed during the reciprocal interaction test. CASPR2-IgG exposed mice spent more time engaged in those activities than HC-IgG exposed mice [U = 19, Z = −2.024, P = 0.04; Fig. 5f, right panel]. In a nest-building test, a species-typical behavior claimed to be potentially relevant to home–cage social interaction [23], CASPR2-IgG exposed mice scored less than the HC-IgG exposed animals [U = 39, Z = −2.126, P = 0.03; Fig. 5h]. All mice were also tested for spontaneous alternation in the T-maze, a test of short-term memory, and a trend towards a decrease in alternation was noted in the CASPR2-IgG exposed mice [t(45) = −1.874, P = 0.067; Fig. 5i]; this is of potential relevance considering that Cntnap2 knockout mice were found to alternate less in the same test [23].

The results from all behavior tests were first analyzed without separating the genders. On further analysis (Online Resource 8), we found no gender effect in any of the behavioral parameters analyzed, with the exception of the nesting test [H(3) = 9.6, P = 0.02; Kruskal–Wallis test], which showed lower scores in CASPR2-IgG exposed males (U = 33, P = 0.01; Mann–Whitney test) but not in females (U = 18, P = 1.0; Mann–Whitney test). A trend towards a gender effect was also noted for the light–dark test and the time spent in non-social activities during the reciprocal social interaction test, with male CASPR2-IgG mice being more affected.

At 12 months, coded brains from 3 to 6 animals in each group were randomly selected for histological analysis. In particular, since abnormalities of neuronal migration were reported in this brain region in the Cntnap2 knockout model [23], the somatosensory cortex was evaluated for the presence of alterations in normal cortical structure. No gross morphological abnormalities and no morphometric differences were observed in the overall thickness of the cortex or in the thickness of the upper, middle, and deep layers, as assessed by Nissl staining (Fig. 6a).

Laminar position patterns, analyzed by assessing FOXP2-positive (marker of layer VI) neurons, did not reveal any differences between CASPR2-IgG and HC-IgG exposed groups (data not shown). However, there was an increase in the density of CUX1-positive cells (marker of layers II–IV) in layers V/VI in CASPR2-IgG exposed animals [t(4) = 3.63, P = 0.02], suggesting a neuronal migration defect (Fig. 6b).

To determine which neurons were expressing CUX1 in layers V/VI, the GABAergic and glutamatergic markers GAD65/67 and glutaminase, respectively, were used. In both CASPR2-IgG and HC-IgG exposed animals, most CUX1-positive neurons in layers V/VI were glutamatergic and none were identified as GABAergic (Fig. 6c).

There were no morphometric differences between groups with respect to the prefrontal cortices (anterior cingulate, prelimbic, and infralimbic cortices), corpus callosum, hippocampus or cerebellum, and no gross morphological abnormalities or neuronal ectopias/misalignments were detected in any of these brain areas (Online Resource 9 to 13).

CD68/Iba1-positive cells (defining activated microglia) were quantified in the prelimbic, infralimbic, and somatosensory cortices. All P values below are adjusted according to the Holm–Sidak correction for multiple comparisons. There was an average 16% increase in mean Iba1/CD68-positive microglial densities in the CASPR2-IgG exposed animals, across all layers of the studied areas (Fig. 7a, b). In the prelimbic cortex, there was an increase in CASPR2-IgG exposed mice in all layers [layer I: t(12) = 2.2, P = 0.049; layers II–IV: t(12) = 4.3; P = 0.0032, layer V–VI: t(12) = 4.1; P = 0.0032), as also in the infralimbic cortex [layer I: t(12) = 3.5, P = 0.0095; layers II–III: t(12) = 3.0, P = 0.01; layers V–VI: t(12) = 5.2, P = 0.0007]. In the somatosensory cortex, the same trend was observed in all layers, statistically significant in layers I and II–IV [layer I: t(12) = 3.4, P = 0.014; layers II–IV: t(12) = 3.4, P = 0.014; layer V–VI: t(12) = 2.1, P = 0.054]. The results in embryos at E18.5 (Exp 2) were variable but showed a similar trend towards increased CD68-positive microglia (P = 0.01 uncorrected; Online Resource 14).

A detailed morphological analysis of parameters of microglial activation (number of ramifications and soma size) was performed in IBA-1-positive cells from the somatosensory cortex in both groups (n = 101 cells/group; 30–40 cells/animal; 3 animals/group) (Fig. 7c). Microglia in CASPR2-IgG exposed animals were significantly less ramified (U = 3.8; P = 0.001) and had increased soma size (U = 6.2; P = 0.014), consistent with an activated phenotype (Fig. 7d).

It is well established that microglia play a role in synaptic pruning [22]. We identified the post-synaptic density of glutamatergic synapses using PSD95, confirmed by its association with the presynaptic terminal marker synaptophysin (Online Resource 15), and used a robust stereological sampling method to provide a large number of PSD95-positive profiles per layer (>2000 profiles). Our method covered a sufficient volume of each cortical layer to ensure sufficient sampling of the irregularly spaced PSD95-positive puncta in the neuropil and thus provided an accurate assessment of mean synaptic puncta density. We found a 15–52% reduction in the number of PSD95-positive synaptic profiles across the prelimbic, infralimbic, and somatosensory cortices in the CASPR2-IgG exposed animals (Fig. 8a, b). In the prelimbic cortex, the PSD95-positive synaptic profiles were reduced in layers I and II–III [layer I: t(12) = 7.0, P = 0.00004; layers II–III: t(12) = 4.9, P = 0.0007], with a strong trend in the deep layers [layer V–VI: t(12) = 2.1, P = 0.055]. In the infralimbic cortex, a significant decrease was seen in all layers [layer I: t(12) = 5.1, P = 0.0005; layers II–III t(12) = 7.3, P = 0.00003; layer V–VI t(12) = 4.1, P = 0.001], and also in the somatosensory cortex [layer I: t(12) = 3, P = 0.02; layers II–IV: t(12) = 4.5, P = 0.002; layer V–VI: t(12) = 2.2, P = 0.049]. These results demonstrate increased microglia and reduced glutamatergic synaptic profiles in the CASPR2-IgG exposed mice that were apparent as late as 12 months after birth.

Consistent with a decrease in glutamatergic synaptic profiles, CASPR2-IgG exposed mice had a significant reduction in the density of GluA1 profiles in the somatosensory cortex [t(12) = 4.2; P = 0.0036]. Both synaptic (GluA1/PSD95-positive puncta) and extra-synaptic (GluA1-positive/PSD95-negative puncta) GluA1 were numerically reduced in the CASPR2-IgG exposed mice, but this difference was significant only for extra-synaptic GluA1 profiles [t(12) = 2.9; P = 0.028] (Fig. 8 c, d). GluA1 expression was also examined in the embryos at E18.5, but, at this stage, no differences in total GluA1 expression were found between groups by western blotting (Online Resource 16).

Despite the reduction in synaptic profiles, the numbers of NeuN-positive neurons in the medial prefrontal cortex (prelimbic and infralimbic cortices) were not different between CASPR2-IgG and HC-IgG exposed animals (Fig. 8e, f). In the somatosensory cortex, there was a modest decrease in the neuronal densities in layer II–IV (P = 0.02), but not in layer I (P = 0.8) or layer V–VI (P = 0.1).