Therapeutic testosterone administration preserves excitatory synaptic transmission in the hippocampus during autoimmune demyelinating disease

Abstract

Over 50% of multiple sclerosis (MS) patients experience cognitive deficits, and hippocampal-dependent memory impairment has been reported in >30% of these patients. While postmortem pathology studies and in vivo magnetic resonance imaging demonstrate that the hippocampus is targeted in MS, the neuropathology underlying hippocampal dysfunction remains unknown. Furthermore, there are no treatments available to date to effectively prevent neurodegeneration and associated cognitive dysfunction in MS. We have recently demonstrated that the hippocampus is also targeted in experimental autoimmune encephalomyelitis (EAE), the most widely used animal model of MS. The objective of this study was to assess whether a candidate treatment (testosterone) could prevent hippocampal synaptic dysfunction and underlying pathology when administered in either a preventative or a therapeutic (postdisease induction) manner. Electrophysiological studies revealed impairments in basal excitatory synaptic transmission that involved both AMPA receptor-mediated changes in synaptic currents, and faster decay rates of NMDA receptor-mediated currents in mice with EAE. Neuropathology revealed atrophy of the pyramidal and dendritic layers of hippocampal CA1, decreased presynaptic (Synapsin-1) and postsynaptic (postsynaptic density 95; PSD-95) staining, diffuse demyelination, and microglial activation. Testosterone treatment administered either before or after disease induction restores excitatory synaptic transmission as well as presynaptic and postsynaptic protein levels within the hippocampus. Furthermore, cross-modality correlations demonstrate that fluctuations in EPSPs are significantly correlated to changes in postsynaptic protein levels and suggest that PSD-95 is a neuropathological substrate to impaired synaptic transmission in the hippocampus during EAE. This is the first report demonstrating that testosterone is a viable therapeutic treatment option that can restore both hippocampal function and disease-associated pathology that occur during autoimmune disease.

Figures

Figure 1.

Experimental design and standard clinical scores in EAE. A, Experimental design depicting the timing of gonadectomy (Day −14, denoted by x), placebo or testosterone pellet implantation (day −7, denoted by circles), EAE induction (day 0 and 7, denoted by arrows), daily clinical scoring (day 7–45, denoted by dashed line), and electrophysiology (day 21–45, denoted by gray line). Pertussis injections (intraperitoneal, i.p.) were given to both EAE groups on day 0 and 2, as part of standard EAE induction (data not shown). All mice were age-matched C57BL/6 adult males that were selected for electrophysiological recording in an intermixed fashion (one per day), so that mice from each experimental group were measured every 3 d. Brain tissue was separated depending on type of study: right hemispheres were used for electrophysiology; respective left hemispheres were prepared for pathology studies. All pathology experiments were conducted once all left hemisphere tissue from mice in all conditions had been collected. B, EAE clinical scores were recorded in placebo-treated (PLAC + EAE, red) or testosterone-treated (T + EAE, blue) mice as well as in non-EAE-induced healthy controls (NL, black). Placebo-treated mice with EAE exhibited a moderately severe clinical course, while testosterone-treated mice exhibited significantly reduced clinical severity. It is important to note that statistical variance was not altered as mice were individually selected for electrophysiological recording. Data are representative of two separate experiments. Repeated-measures ANOVA with post hoc pairwise comparisons revealed that PLAC + EAE was significantly different from other two groups, p < 0.05, n = 5 mice per group.

Figure 2.

Hippocampal atrophy occurs during EAE and testosterone treatment prevents this. A–C, Representative hippocampal sections stained with Nissl from normal (NL; A), placebo-treated EAE (PLAC + EAE; B), and testosterone-treated EAE (T + EAE; C) mice depict 4× magnification of the various hippocampal subregions: CA1 pyramidal layer (pyr), CA1 stratum oriens (so), CA1 stratum radiatum (sr), CA2/3, dentate gyrus (DG), and corpus callosum (CC). D–F, The CA1 pyramidal layer shown at 40× magnification, had significantly reduced volume in placebo-treated EAE mice (E), compared with healthy controls (D), quantification in G. With testosterone treatment during EAE (F), the CA1 pyr volume was preserved to levels similar to those in controls (D and G). H, CA1 stratum radiatum (CA1 sr) area was also significantly reduced in placebo-treated EAE mice compared with healthy controls, and this atrophy was prevented in testosterone-treated EAE mice. I, Alternatively, stratum oriens (CA1 so) area was not significantly changed with EAE or testosterone treatment of EAE, compared with healthy controls. J, CA3 area was significantly reduced in placebo-treated mice with EAE, compared with healthy controls, and preserved in testosterone-treated mice with EAE. Estimated CA1 pyr volume (mm3) and CA1 sr, CA1 so, and CA3 area (mm2) depict representative means normalized to mean area and volume of healthy controls (NL). One-way ANOVA and Newman–Keuls post hoc analysis revealed significant difference between three groups. *p = 0.05, n = 3 mice per group (5 sections per mouse). Scale bars, 20 μm.

Figure 3.

Synaptic staining in the hippocampus is disrupted in EAE and preserved with testosterone treatment. A–I, Fluorescent images depict representative hippocampal sections from healthy (A–C), placebo-treated EAE (D–F), and testosterone-treated EAE (G–I) mice, where Synapsin-1 (Syn-1; Cy3-green), PSD-95 (Cy5-red), and DAPI (blue) staining are shown at 60× magnification. During EAE, Syn-1+ puncta were significantly decreased in the CA1 region of the hippocampus (D), compared with healthy controls (A). This decrease was prevented with testosterone treatment during EAE (G), quantified in J. Similarly, PSD-95 was also significantly decreased in placebo-treated EAE mice, and preserved in testosterone-treated EAE mice (B, E, H, respectively, and quantified in K). C, F, I, Merged images of both presynaptic (Syn-1) and postsynaptic (PSD-95) staining in selected areas (dashed square) of CA1 sr demonstrate the extent of colocalization within CA1 dendrites. One-way ANOVA and Newman–Keuls post hoc analysis revealed statistical significance in presynaptic and postsynaptic integrity where *p = 0.04, n = 3 mice per group, and **p < 0.001, n = 3 mice per group. Scale bars, 10 μm.

Figure 4.

Excitatory synaptic transmission is impaired in EAE, and testosterone treatment prevents this. Input/output relationships were measured by electrophysiology. A, When comparing fiber volley amplitudes ranging between 1.2 and 2.0 mV, maximal fEPSP slopes were significantly lower in EAE (PLAC + EAE, red, n = 10 slices from 3 animals) compared with normal (NL, black, n = 10 slices from 4 animals). With testosterone treatment during EAE (T + EAE, blue, n = 12 slices from 5 animals) increased fiber volley amplitudes were attained (2.0 mV and greater), and fEPSP responses were similar to those of normal controls. B, When maximal fEPSPs were compared across all groups, placebo-treated EAE mice had significantly lower responses as compared with healthy controls, while maximal fEPSPs in testosterone-treated EAE mice were not significantly different from controls. Individual datasets were compared using Student's t test, where * indicated groups significantly different, p = 0.04. C, Sample waveforms depict representative input/output responses recorded from animals in each experimental condition. Each set of waveforms shows fEPSP responses 25, 50, and 75% of maximal fEPSP slope in gray, while each maximal fEPSP slope (100% response) is color coded to experimental condition (black, NL; red, PLAC + EAE; blue, T + EAE). D, Pairs of presynaptic fiber stimulation pulses were delivered with interpulse intervals of 25 ms, 50 ms, 100 ms, 200 ms, or 275 ms). PPF was unchanged in the hippocampal CA1 region of placebo-treated EAE mice (n = 7 slices from 3 animals) and testosterone-treated EAE mice (n = 8 slices from 5 animals), compared with healthy control mice (n = 6 slices from 4 animals).

Figure 5.

AMPAR-mediated synaptic current frequency and NMDAR-mediated synaptic current decay rate are reduced during EAE. A, mEPSCs were recorded at −70 mV in the presence of 1.0 μm TTX and 100 μm picrotoxin. B, mEPSC amplitude cumulative probability distribution was not significantly different for cells from healthy control mice (NL, black, n = 17 cells from 5 mice) and placebo-treated EAE mice (PLAC + EAE, red, n = 9 cells from 4 mice). C, The interevent interval cumulative probability distribution in cells from placebo-treated EAE (PLAC + EAE, red) mice was shifted right, compared with healthy control (NL, black) mice, indicating longer interevent intervals between mEPSCs. D, Mean mEPSC amplitude was not considerably altered in cells from placebo- or testosterone-treated mice with EAE (T + EAE, blue, n = 13 cells from 4 mice), compared with healthy control mice. E, mEPSC frequency was strongly reduced, however, in cells from placebo-treated EAE mice and testosterone-treated EAE mice compared with mEPSC frequency in cells from healthy control mice, where *p < 0.05, one-way ANOVA followed by Dunnet's test comparisons to control. F, Examples of evoked EPSCs recorded at holding potentials of −80 mV and +40 mV in CA1 pyramidal cells from healthy control (NL, black), placebo-treated EAE (PLAC + EAE, red), and testosterone-treated EAE (T + EAE, blue) mice. G, NMDA receptor/AMPA receptor ratios of evoked EPSCs recorded at either −80 mV or +40 mV are not altered in EAE. H, The decay of NMDA receptor-mediated EPSCs recorded at +40 mV was significantly faster in cells from placebo-treated EAE mice (PLAC + EAE, red, n = 11 cells from 4 mice) compared with healthy control mice (NL, black, n = 12 cells from 5 mice). Interestingly, NMDAR-mediated synaptic current decay rate was not significantly different in testosterone-treated EAE mice (T + EAE, blue, n = 12 cells from 4 mice), compared with healthy control mice, where *p < 0.05, one-way ANOVA followed by Dunnet's test comparisons to control.

Figure 6.

PSD-95 levels are strongly correlated to maximal postsynaptic responses. Linear regression analyses were performed to determine potential relationships between pathology and electrophysiological response. A, PSD-95% area was plotted in the x-axis, while maximal fEPSP slopes corresponding to each animal were plotted on the y-axis. When Pearson's correlation test was calculated across conditions, the correlation coefficient was large, ρ = 0.7627, indicating a strong linear relationship between PSD-95 and fEPSP slope, p = 0.0168, n = 9 mice total. B, Synapsin-1, however, was not significantly correlated to fiber volley amplitude slope, as ρ = 0. 2581, p = 0.5025, n = 9 mice, when compared across all conditions.

Figure 7.

Decreased MBP immunoreactivity occurs in the hippocampal CA1 regions of mice with EAE and is preserved with testosterone treatment. A–C, Representative fluorescent images depict MBP immunoreactivity (Cy5-red) in the stratum oriens (so) region of CA1 within healthy (A), placebo-treated EAE (B), and testosterone-treated EAE (C) mice at 40× magnification. EAE caused a significant reduction in myelin staining within the CA1 region of the hippocampus in placebo-treated mice (B), compared with myelin staining in hippocampus of healthy control mice (A, D). In testosterone-treated EAE mice, myelin levels were similar to those found in healthy controls (C). Analysis was conducted on 10× magnification images where MBP percentage area represents total MBP immunoreactivity as a percentage of total CA1 area imaged. One-way ANOVA revealed significant differences in myelin staining across conditions, *p < 0.05, n = 3 per group, and Newman–Keuls post hoc tests indicated that placebo-treated EAE mice were significantly different from both other groups. E–G, Additional magnified images are depicted to verify specificity of MBP stain within each representative hippocampal section, taken from A–C, dashed white squares and rotated clockwise 90°. Here, MBP (red) colocalizes with NF-200 (Cy3-green) to create a bright yellow fluorescence (indicated by arrowheads) which is more often observed in the hippocampal CA1 so of healthy control mice (E), and testosterone-treated EAE mice (G), but not commonly seen in the hippocampal CA1 so of placebo-treated EAE mice (F). Scale bars, 10 μm.

Figure 8.

Microglial activation was significantly elevated in the hippocampus during EAE, and not during testosterone treatment of EAE. A–C, Fluorescent images depicted here are 10× magnified images representative of hippocampal sections from healthy control mice (A), placebo-treated EAE mice (B), and testosterone-treated EAE mice (C), where cells of microglial/macrophage lineage are stained with Iba1 (Cy3-green) and DAPI (blue). Iba1+ cells with morphology characteristic of reactive microglia were relatively low in healthy controls (A), significantly increased in placebo-treated EAE mice (B), and reduced to levels similar to normal in testosterone-treated EAE mice (C). The number of Iba1+ cells per mm3 were counted and presented as whole numbers (D). E–G, Representative images of microglia (Iba-1, Cy3-green) and neuronal perikarya (NeuN, Cy5-red) of CA1 pyramidale (pyr) layer within the hippocampus of animals from each condition, shown here at 40× magnification, demonstrate that some cells with morphology indicative of reactive microglia associate with pyramidal neurons of CA1, and more so in placebo-treated EAE mice (F). One-way ANOVA revealed significant differences in microglia activation, *p < 0.05, n = 3 per group, and Newman–Keuls post hoc tests showed that placebo-treated EAE mice (PLAC + EAE) were different from mice in other two groups. Scale bars, 10 μm.

Figure 9.

DHT is not capable of ameliorating EAE clinical severity or preventing CA1 atrophy. A, DHT-treated mice with EAE (DHT + EAE, green circles, n = 5 mice) demonstrated clinical scores similar to placebo-treated mice with EAE (PLAC +EAE, red triangles, n = 5 mice), which contrasted from testosterone-treated mice with EAE (T + EAE, blue triangles, n = 5 mice) that had clinical scores ameliorated and not different from placebo-treated healthy control mice (NL, black squares, n = 5 mice). B, When CA1 pyramidal (pyr) layer volume was compared across conditions, DHT-treated mice with EAE (n = 5 mice) had significantly reduced volumes compared with normal healthy controls (n = 35 mice), and DHT animals were not different from placebo-treated mice with EAE (n = 5 mice). This finding is in direct contrast to the effect testosterone had on CA1 pyr layer volume (T + EAE, n = 5 mice), which was to preserve CA1 pyr layer volume to levels similar to healthy control animals. One-way ANOVA demonstrated an effect of condition on EAE clinical score (p = 0.0131, n = 5 mice per group). Newman–Keuls multiple-comparisons post hoc analysis indicated that placebo-treated mice with EAE and DHT-treated mice with EAE both differed in CA1 pyr volume compared with healthy control animals and testosterone-treated mice with EAE, *p < 0.05, n = 5 mice per group.

Figure 10.

Testosterone treatment after EAE induction restores synaptic transmission and corresponding synaptic protein levels within the hippocampus during EAE. A, Experimental design highlighting testosterone (or placebo) treatment after disease induction (day 10) when standard EAE signs first appeared. B, In contrast to pretreatment (Fig. 1), treatment after disease induction did not decrease standard EAE scores until ∼3 weeks after it was initiated, a time point later in disease (day 35) when comparing testosterone-treated EAE (EAE + T, blue, n = 5 mice) with placebo-treated EAE (EAE + PLAC, red, n = 5 mice). C, Maximal fEPSP slopes were reduced in placebo-treated mice with EAE (EAE + PLAC, red, n = 21 slices from 7 mice), but restored in testosterone-treated EAE mice (EAE + T, blue, n = 24 slices from 8 mice) to levels similar to healthy controls (NL, black, n = 15 slices from 5 mice); *p < 0.05, one-way ANOVA with Newman–Keuls multiple comparisons. D, PSD-95% area and Syn-1+ puncta were significantly reduced in EAE mice that received placebo treatment after disease induction (iii and iv, respectively, n = 7 mice) compared with healthy control mice (i and ii, n = 5 mice), but testosterone treatment after EAE induction restored both synaptic protein levels (v and vi, graphs vii and viii, n = 8 mice). One-way ANOVA with Newman–Keuls multiple-comparisons analyses revealed significance where *p < 0.05, **p < 0.01. E, Cross-modality correlation analyses revealed that testosterone-mediated effects on synaptic transmission (maximal fEPSP slope) were significantly correlated to effects on PSD-95 synaptic protein levels, where Pearson's correlation test indicated a strong linear relationship between PSD-95 and fEPSP slope, ρ = 0.63898 p = 0.0032, n = 20 mice total. F, Syn-1+ puncta were not significantly correlated to fiber volley amplitude slope, as ρ = 0.0608, p = 0.8045, n = 20 mice total, when compared across all conditions.