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. 2018 Jun;59(6):1257-1268.
doi: 10.1111/epi.14429. Epub 2018 May 25.

Semaphorin 4D promotes inhibitory synapse formation and suppresses seizures in vivo

Daniel W M Acker  1 , Irene Wong  1 , Mihwa Kang  1 , Suzanne Paradis  1
Affiliations

Semaphorin 4D promotes inhibitory synapse formation and suppresses seizures in vivo

Daniel W M Acker et al. Epilepsia. 2018 Jun.

Abstract

Objective: We previously discovered a role for the extracellular domain of the transmembrane protein semaphorin 4D (Sema4D) as a fast-acting, selective, and positive regulator of functional γ-aminobutyric acid (GABA)ergic synapse formation in hippocampal neuronal culture. We also demonstrated that Sema4D treatment increases inhibitory tone and suppresses hyperexcitability in an organotypic hippocampal slice culture model of epilepsy. Here, we investigate the ability of Sema4D to promote GABAergic synapse formation and suppress seizure activity in vivo in adult mice.

Methods: We performed a 3-hour, intrahippocampal infusion of Sema4D or control protein into the CA1 region of adult mice. To quantify GABAergic presynaptic bouton density, we performed immunohistochemistry on hippocampal tissue sections isolated from these animals using an antibody that specifically recognizes the glutamic acid decarboxylase isoform 65 protein (GAD65), which is localized to presynaptic GABAergic boutons. To assess seizure activity, we employed 2 in vivo mouse models of epilepsy, intravenous (iv) pentylenetetrazol (PTZ) and hippocampal electrical kindling, in the presence or absence of Sema4D treatment. We monitored seizure activity by behavioral observation or electroencephalography (EEG). To assay the persistence of the Sema4D effect, we monitored seizure activity and measured the density of GAD65-positive presynaptic boutons 3 or 48 hours after Sema4D infusion.

Results: Sema4D-treated mice displayed an elevated density of GABAergic presynaptic boutons juxtaposed to hippocampal pyramidal neuron cell bodies, consistent with the hypothesis that Sema4D promotes the formation of new inhibitory synapses in vivo. In addition, Sema4D acutely suppressed seizures in both the PTZ and electrical kindling models. When we introduced a 48-hour gap between Sema4D treatment and the seizure stimulus, seizure activity was indistinguishable from controls. Moreover, immunohistochemistry on brain sections or hippocampal slices isolated 3 hours, but not 48 hours, after Sema4D treatment displayed an increase in GABAergic bouton density, demonstrating temporal correlation between the effects of Sema4D on seizures and GABAergic synaptic components.

Significance: Our findings suggest a novel approach to treating acute seizures: harnessing synaptogenic molecules to enhance connectivity in the inhibitory network.

Keywords: GABAergic; Sema4D; epilepsy; hippocampus; synaptogenesis.

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Conflict of interest statement

Competing interests

Author Suzanne Paradis has submitted Provisional US Patent Application No. 61/756,809 entitled "Methods of Modulating GABAergic Inhibitory Synapse Formation and Function Using Sema4D." Co-inventors: Kuzirian, Marissa; Moore, Anna; Paradis, Suzanne. The remaining authors have no conflicts of interest.

Figures

Fig. 1
Fig. 1. Sema4D treatment increases GABAergic bouton density in vivo
Mice (C57Bl/6, male, P42–P59) were anesthetized and treated with Sema4D or vehicle solution by unilateral intra-hippocampal infusion over 10 minutes. The mice were sacrificed 3 hours following treatment. (A) Coronal sections through mouse hippocampus contralateral (top) and ipsilateral (bottom) to the infusion site. The infusion is marked by dextran-488 (green), and nuclei are labelled by DAPI (blue). White boxes denote regions of interest from which cells analyzed as in C are selected. Scale bar represents 200 µm. (B) CA1 principal layer cells in coronal sections through Sema4D or vehicle treated brain hemispheres. Sections are stained with an antibody that specifically recognizes GAD65 (red) and with DAPI (blue). Scale bar represents 10 µm. (C) Density of GAD65 puncta on the perimeter of CA1 principal cell layer somas. In all cases, ipsilateral is the site of infusion and contralateral was not treated. Colored points represent individual cells and black points represent means for individual animals; black lines connecting dots indicate the same animal. Boxplots summarize individual cell data. In this and all figures: in boxplots, hinges represent the first and third quartile. Statistics were performed on delta scores calculated within animal by subtracting the mean bouton density on the contralateral side from the mean bouton density on the ipsilateral side. Delta scores were significantly greater in Sema4D-treated mice than in vehicle-treated mice (p<0.01, unpaired, two-tailed t test). n=8 mice and ≥174 cells per condition.
Fig. 2
Fig. 2. Sema4D treatment increases i.v. PTZ-induced seizure thresholds
(A) Timeline of the experiment. Mice (C57Bl/6, male, P50–P80) were implanted unilaterally with cannulas targeting CA1, then given 5–10 days to recover. After recovery, a 3-hour intra-hippocampal Sema4D or vehicle treatment was administered. Immediately following treatment, PTZ was infused through the tail vein for up to 5 min. (B) PTZ dose (d=tcr/w, where d is the PTZ dose received, t is the time since the start of infusion, c is the concentration of PTZ, r is the rate of infusion, and w is the weight of the animal) versus seizure class (see methods for description of PTZ seizure classes). There was a significant main effect of treatment on dose, p<0.01, mixed-design ANOVA on ranks. There was no significant interaction between treatment and seizure class. n=9 mice per condition.
Fig. 3
Fig. 3. The anti-seizure and increased GABAergic density-promoting effects of Sema4D are temporally correlated
(A) Timeline of the experiment shown in B. Mice (C57Bl/6, male, P50–P80) were implanted unilaterally with cannulas targeting CA1, then given 5–10 days to recover. After recovery, an intra-hippocampal Sema4D or vehicle treatment was administered. Forty-eight hours after treatment, PTZ was infused through the tail vein for up to 5 min. (B) PTZ dose versus seizure class. There was no significant main effect of treatment on dose, and there was no significant interaction between treatment and seizure class. Mixed-design ANOVA on ranks; n=11 vehicle-treated and n=7 Sema4D-treated mice. (C, D) Mice (C57Bl/6, male, P49–P59) were anesthetized and treated with Sema4D or vehicle solution by unilateral, intra-hippocampal infusion over 10 minutes. The mice were sacrificed 3 hours (n=4 mice, all Sema4D-treated) or 48 hours (n=6 mice, vehicle-treated and n=5 mice, Sema4D-treated) following treatment. (C) CA1 principal layer cells in coronal sections through Sema4D or vehicle treated brains. Sections are stained with an antibody that specifically recognizes GAD65 (red) and with DAPI (blue). Scale bar represents 10 µm. (D) Density of GAD65 puncta on the perimeter of CA1 principal cell layer somas. Colored points represent individual cells and black points represent means for individual animals; black lines connecting dots indicate the same animal. Boxplots summarize individual-cell data. In all cases, ipsilateral is the site of infusion and contralateral was not treated. Statistics were performed by ANOVA with post-hoc, Bonferroni-corrected, unpaired, two-tailed, t tests on delta scores calculated within animal by subtracting the mean bouton density on the contralateral side from the mean bouton density on the ipsilateral side. Delta scores were significantly greater in Sema4D-treated mice sacrificed 3 hours after treatment than mice of either treatment group sacrificed 48 hours after treatment (p<0.05). Delta scores did not differ between Sema4D and vehicle-treated mice sacrificed 48 hours after treatment (p>0.05).
Fig. 4
Fig. 4. Acute Sema4D treatment protects against electrically kindled seizures
(A) Timeline of the experiment. Mice (C57Bl/6, male, P56–P80) were implanted bilaterally with cannulas and electrodes targeting DG, then given 5–10 days to recover. The rapid kindling protocol (see methods) was conducted over a three-day period (yellow triangles denote periods of stimulus delivery). Kindling was followed by a 48-hour rest period, then a baseline evoked seizure test (pre-test), 3 hours of Sema4D or vehicle treatment, and a post-treatment evoked seizure test (post-test). Another post-test was administered 48-hours post-treatment (+48hrs). (B) Maximal behavioral seizure score per kindling stimulus versus experimental day (see methods for description of Racine seizure scores). *p<0.05, ***p<0.001, unpaired two-tailed t tests with Bonferroni corrections. Error bars represent SEM. (C) Delta (post-test minus pre-test or +48hrs minus pre-test) mean seizure scores for post-test and +48hrs. **p<0.01, mixed-design ANOVA on ranks with post-hoc unpaired two-tailed t tests with Bonferroni corrections. (D) Delta (calculated as described above) time spent in kindled motor seizures (KMS) for post-test and +48hrs. **p<0.01, mixed-design ANOVA on ranks with post-hoc unpaired two-tailed t tests with Bonferroni corrections. (E) Total EEG power (0.5–30Hz) versus time during the evoked seizure tests. Recordings were contralateral to the site of stimulation. Yellow bars denote the time of stimulus presentation. Error bars indicate SEM. (F) Delta (calculated as described above) after-discharge EEG power for post-test and +48hrs. *p<0.05, mixed-design ANOVA on ranks with post-hoc unpaired two-tailed t tests with Bonferroni corrections. n=9 mice per condition (all panels).
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