Estriol preserves synaptic transmission in the hippocampus during autoimmune demyelinating disease

Abstract

Cognitive deficits occur in over half of multiple sclerosis patients, with hippocampal-dependent learning and memory commonly impaired. Data from in vivo MRI and post-mortem studies in MS indicate that the hippocampus is targeted. However, the relationship between structural pathology and dysfunction of the hippocampus in MS remains unclear. Hippocampal neuropathology also occurs in experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. Although estrogen treatment of EAE has been shown to be anti-inflammatory and neuroprotective in the spinal cord, it is unknown if estrogen treatment may prevent hippocampal pathology and dysfunction. In the current study we examined excitatory synaptic transmission during EAE and focused on pathological changes in synaptic protein complexes known to orchestrate functional synaptic transmission in the hippocampus. We then determined if estriol, a candidate hormone treatment, was capable of preventing functional changes in synaptic transmission and corresponding hippocampal synaptic pathology. Electrophysiological studies revealed altered excitatory synaptic transmission and paired-pulse facilitation (PPF) during EAE. Neuropathological experiments demonstrated that there were decreased levels of pre- and post-synaptic proteins in the hippocampus, diffuse loss of myelin staining and atrophy of the pyramidal layers of hippocampal cornu ammonis 1 (CA1). Estriol treatment prevented decreases in excitatory synaptic transmission and lessened the effect of EAE on PPF. In addition, estriol treatment prevented several neuropathological alterations that occurred in the hippocampus during EAE. Cross-modality correlations revealed that deficits in excitatory synaptic transmission were significantly correlated with reductions in trans-synaptic protein binding partners known to modulate excitatory synaptic transmission. To our knowledge, this is the first report describing a functional correlate to hippocampal neuropathology in any MS model. Furthermore, a treatment was identified that prevented both deficits in synaptic function and hippocampal neuropathology.

Figures

Figure 1. Experimental design and standard clinical scores in EAE

(A) Experimental design depicting the timing of placebo or estriol pellet implantation (D -7), EAE induction (D 0 & D 7), daily clinical scoring (D 7-40) and electrophysiology (D 21-40). Pertussis injections (intraperitoneal, i.p.) were given to both EAE groups on D0 and D2, as part of standard EAE induction, not shown. All mice were age-matched C57Bl/6 adult females, and were uniform randomly selected (one per day) for electrophysiological recording. On each day of electrophysiology, the right hemisphere was prepared for electrophysiology, while the left hemisphere of each mouse was prepared for pathology studies and kept until all tissue from all conditions had been collected. (B) EAE clinical scores were recorded in placebo-treated (EAE + PLAC, black star, dashed black line) or estriol-treated (EAE + ESTRIOL, gray triangle, dashed gray line) mice as well as in non-EAE induced healthy controls (NL, black square, solid black line). Placebo-treated mice with EAE exhibited a severe clinical disease course, while estriol-treated mice exhibited significantly reduced clinical severity. Data are representative of two separate experiments, and experiment has been repeated more four times. Repeated measures ANOVA with post-hoc pair-wise comparisons, revealed that placebo-treated EAE mice were significantly different from other two groups beginning at D12 of disease, * p < 0.05, n = 5 per group per experiment.

Figure 2. Excitatory synaptic transmission and paired-pulse facilitation are altered during EAE

(A) Input/output (IO) curves were created by comparing fiber volley amplitude (FV Amp; mV) to field excitatory postsynaptic potential (fEPSP; mV/ms) during four different stimulation intensities of presynaptic fiber stimulation that caused fEPSP responses of 25, 50, 75 and 100% maximal fEPSP amplitude. One-way ANOVA analyses demonstrated that placebo-treated EAE (EAE+PLAC; black star, dashed black line) fEPSP responses were significantly reduced at each point along the IO curve (percentage of maximal fEPSP slope: 25, 50 75 and 100%) in comparison to healthy controls (NL; black squares, solid black line), p

Figure 3. Hippocampal PSD-95 levels are decreased in EAE and preserved with estriol treatment

(A-F) Fluorescent images depict the stratum radiatum with the CA1 region of representative hippocampal slices from healthy (panel A-B), placebo-treated EAE (panel C-D) and estriol-treated EAE (panel E-F) mice, where PSD-95 (A, C & E; Cy5-red), and Synapsin-1 (B, D & F; Syn-1; Cy3-green) staining are shown at 60x magnification. During EAE, PSD-95 % area was significantly decreased in the CA1 region of the hippocampus, (C), compared to healthy controls (A). This decrease was prevented with estriol treatment during EAE, (E), quantified in graph G. One-way ANOVA and Bonferroni post-hoc analysis revealed statistical significance in PSD-95 % area where, * indicates p = 0.0324, n = 5 per group. Average number of presynaptic Syn-1+ puncta within the CA1 was not significantly different across conditions. These experiments were repeated twice with similar results. Scale bar 10um.

Figure 4. Presynaptic β-neurexin II and postsynaptic neuroligin-1 are decreased during EAE, but preserved with estriol treatment

Synaptic puncta were measured in the hippocampal CA1 region by counting immunoreactive NRXIIβ puncta (A-B) and NLG1 puncta (C-D) in healthy control, (panels i-ii), placebo-treated EAE, (panels iii-iv), and estriol-treated EAE mice (panels v-vi), respectively. Pseudo-colored confocal images depict synaptic staining (either NRXIIβ or NLG1, Cy3-red) in hippocampus at 60x magnification, while black and white images show outlined puncta from respective confocal images. One-way ANOVA revealed that groups differed significantly in quantity of NRXIIβ puncta, (p = 0.005) and Bonferroni post-hoc test indicated that placebo-treated EAE mice (EAE+PLAC, n = 5 mice) had significantly decreased (* p < 0.01) numbers of NRXIIβ puncta compared to healthy control (NL, n = 5 mice) and estriol-treated EAE mice (EAE+ESTRIOL, n = 4 mice), graph B. Simultaneously, NLG1 puncta quantities were also significantly different across conditions (one-way ANOVA, p = 0.0056) with follow up tests indicating significant difference between healthy controls (NL, n = 5 mice) and placebo-treated EAE mice (EAE+PLAC, ** p < 0.01, n = 5 mice), as well as significant difference between placebo-treated EAE and estriol-treated EAE mice (EAE+ESTRIOL, * p < 0.05, n = 5 mice), graph D. These experiments were repeated twice and similar results were found. Scale bars 10um.

Figure 5. Decreased myelin staining and increased microglial activation occur within the hippocampus during EAE, and estriol treatment preserves myelin staining

(A-C) Pseudo-colored confocal fluorescent images depict myelin basic protein immunoreactivity (MBP, Cy5-red) and cells of microglial/macrophage lineage stained with Iba1 (Cy3-green) and Dapi (blue) in the CA1 region within healthy (A), placebo-treated EAE (B) and estriol-treated EAE (C) mice at 40x magnification. (D) One-way ANOVA revealed significant differences in myelin staining across conditions, p = 0.003, n = 5 mice per group, and Bonferroni post-hoc tests indicated that EAE caused a significant reduction in myelin staining within the CA1 region of the hippocampus in placebo-treated mice (EAE+PLAC, ** p < 0.01, n = 5 mice) compared to myelin staining in hippocampus of healthy control mice (NL, n = 5 mice). Estriol-treated EAE mice (EAE+ESTRIOL, n = 5 mice) had myelin levels significantly increased compared to placebo-treated EAE mice (* p < 0.05), but were still significantly different from myelin staining levels in healthy controls. Analysis was conducted on 10x magnification images where MBP % area represents total MBP immunoreactivity as a percent of total delineated CA1 area imaged. Iba1+ cells with morphology characteristic of reactive microglia were relatively low in healthy controls (A), and increased in placebo-treated EAE mice (B). The number of Iba1+ cells was not significantly different in estriol-treated EAE mice (C), however. The number of Iba1+ cells per mm3 were counted and presented as whole numbers (graph E). Data are representative of two experiments. Scale bar 10um.

Figure 6. Hippocampal CA1 atrophy occurs during EAE and estriol treatment prevents this

(A-C) Nissl-stained hippocampal sections representative of normal (NL; A), placebo-treated EAE (EAE+PLAC; B) and estriol-treated EAE (EAE+ESTRIOL; C) mice depict the CA1 pyramidal layer at 40x magnification. Compared to healthy controls, placebo-treated EAE mice had significantly reduced CA1 pyramidal layer volume, quantified in D. With estriol treatment during EAE, the CA1 pyramidal volume was preserved to levels similar to those in controls. One-way ANOVA and Bonferroni post-hoc analysis revealed significant difference between three groups, (*p = 0.05, n = 5 mice per group), scale bar 10um. This finding has been confirmed by repeated experiments.

Figure 7. Decreased pre- and postsynaptic protein levels are significantly correlated with electrophysiological changes during EAE

Cross-modality correlations were conducted to determine potential neuropathological substrates underlying changes in excitatory synaptic transmission and paired-pulse facilitation during EAE. (A) PSD-95% area was significantly correlated to maximal fEPSP slope throughout experiment, where the correlation coefficient rho = 0.8767, p = 0.019, n = 9 mice total. (B) The average number of presynaptic NRXIIβ puncta was also significantly correlated to postsynaptic responses (fEPSP) where rho = 0.8396, p = 0.0046, n = 9 mice total.