Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Jul 1.
Published in final edited form as: Epilepsy Res. 2023 Apr 27;193:107156. doi: 10.1016/j.eplepsyres.2023.107156

Semaphorin 4D induced inhibitory synaptogenesis decreases epileptiform activity and alters progression to Status Epilepticus in mice

Susannah S Adel 1, Vernon RJ Clarke 1, Aidan Evans-Strong 2, Jamie Maguire 2,3, Suzanne Paradis 1,3
PMCID: PMC10247425  NIHMSID: NIHMS1900984  PMID: 37163910

Abstract

Previously we demonstrated that intra-hippocampal infusion of purified, Semaphorin 4D (Sema4D) extracellular domain (ECD) into the mouse hippocampus rapidly promotes formation of GABAergic synapses and decreases seizure susceptibility in mice. Given the relatively fast action of Sema4D treatment revealed by these studies, we sought to determine the time course of Sema4D treatment on hippocampal network activity using an acute hippocampal slice preparation. We performed long-term extracellular recordings from area CA1 encompassing a 2-hour application of Sema4D and found that hippocampal excitation is suppressed for hours following treatment. We also asked if Sema4D treatment could ameliorate seizures in an acute seizure model: the kainic acid (KA) mouse model. We demonstrate that Sema4D treatment delays and suppresses ictal activity, delays the transition to Status Epilepticus (SE), and lessens the severity of SE. Lastly, we sought to explore alternative methods of Sema4D delivery to hippocampus and thus created an Adeno Associated Virus expressing the ECD of Sema4D. Our data reveal that virally delivered, chronically overexpressed Sema4D-ECD promotes GABAergic synapse formation and suppresses ictal activity and progression to SE. These results provide proof of concept that viral delivery of Sema4D is an efficacious and promising delivery method to abate epileptiform activity and progression to SE.

1. Introduction

Status epilepticus (SE) is a life-threatening neurological emergency characterized by continuous seizure activity lasting greater than 5 minutes (Trinka et al., 2015) which can have serious long-term consequences including neuronal injury and death. First line treatment for SE is intravenous or intramuscular administration of benzodiazepines (BZD; e.g. diazepam) (Cruickshank et al., 2022). Benzodiazepines enhance the activity of GABAA receptor subunits, thereby affecting existing GABAergic synapses, and increasing inhibitory tone in the brain. Unfortunately, ~30% of SE patients do not respond to treatment with BZD plus at least one other anti-seizure medication, resulting in refractory SE (RSE) which has a mortality rate of approximately 35% (Mayer et al., 2002; Shorvon and Ferlisi, 2012) . Thus, the need for innovative anti-seizure medications to treat SE and RSE is dire.

We and others demonstrated that the ECD of Sema4D promotes the stabilization and development of GABAergic synapses on a rapid time scale (~30 minutes) (Acker et al., 2018; Frias et al., 2019; Kuzirian et al., 2013); these synapses become functional within two hours (Kuzirian et al., 2013). We reasoned that because progression to SE occurs within minutes, treatment with the pro-synaptogenic molecule Sema4D could impart therapeutic effects in mouse models of this disease. In addition, our previous studies using in vivo seizure models (intravenous pentylenetetrazol and electrical kindling) demonstrated that acute infusion of the purified Sema4D ECD (Sema4D-Fc) into hippocampus increases GABAergic synapse density, increases time to seizure onset, and decreases seizure severity (Acker et al., 2018). Based on these data we proposed a model whereby Sema4D treatment in hippocampus promotes GABAergic synapse formation, thus increasing inhibitory tone and suppressing seizures (Acker et al., 2018).

In the current study, we addressed three outstanding questions about the effect of Sema4D-dependent synapse formation on brain hyperexcitability using both ex vivo and in vivo preparations. First, we sought to determine the time course of Sema4D treatment on hippocampal circuit function. To accomplish this goal, we used extracellular recordings to assay population spike amplitude in the CA1 region of acute hippocampal slice before, during, and after 2 hours of Sema4D treatment.

Second, we asked if Sema4D protein infusion to the intact hippocampus could impede progression to SE and decrease the severity of seizures. To address this question, we employed KA-induced SE which is an established rodent model of temporal lobe epilepsy that progresses to SE with development of diazepam insensitivity (reviewed in (Reddy and Kuruba, 2013). Thus, we delivered purified Sema4D-Fc protein via cannula to the hippocampus of mice and subjected them to KA-induced SE and subsequent BZD treatment while monitoring seizure severity using EEG.

Third, we sought to determine if chronic delivery of Sema4D protein via viral mediated gene transduction in the hippocampus could suppress seizure onset and severity similar to, or better than, the effect observed with acute infusion of purified Sema4D protein. This line of experimentation also allowed us to test whether we could replace intrahippocampal infusion of Sema4D protein with virus-mediated gene transduction of Sema4D, a more generalizable and tractable delivery method for this potential therapeutic. To answer these questions, we developed and validated adeno associated virus expressing Sema4D-ECD and tested whether chronic Sema4D-ECD expression via viral mediated gene transduction could increase GABAergic synapse density. In addition, we asked if viral delivery of Sema4D to intact hippocampus could suppress progression to and severity of SE using the KA model.

2. Methods

2.1. Animals

C57BL/6 mice and Long-Evans rats were purchased from the Charles River Laboratories and housed in the animal facility at Brandeis University or at Tufts University. Animals were maintained with a 12-hour light-dark cycle. Food and water were available ad libitum. Animal procedures were performed with approval from the Brandeis University Institutional Animal Care and Use Committee and the Tufts University School of Medicine Animal Care and Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals (NRC). Animal studies were performed in compliance with ARRIVE guidelines as described in this methods section.

2.2. Population spike recordings

Brains were isolated from Long-Evans rats of both sexes between 20-25 weeks old. As this experiment was designed to test the effect of Sema4D treatment on circuit excitability only; sex as a biological variable was not part of the experimental design. The brain was rapidly dissected and placed in ice-chilled, oxygenated modified artificial cerebrospinal fluid (aCSF) comprising of (in mM): NaCl 124, KCl 3.7, NaHCO3 24.6, CaCl2 1, MgSO4 3, D-glucose 10, KH2PO4 1.2 saturated with 95% O2 / 5% CO2. Parasagittal slices (400 μm thick) containing the hippocampal region were prepared in ice-chilled, oxygenated aCSF using a Leica VT1200 vibrating microtome (Leica Biosystems Inc.).

Following equilibration for at least an hour, slices were transferred to a brain slice interface chamber (model BSC2, Scientific Systems Design, Inc.); slices rested on filter paper at the interface of the perfusing solution (0.4 ml min−1) which comprised standard aCSF saturated with 95% O2 / 5% CO2. Field recordings were obtained using a glass microelectrode (resistance ~ 2-4MΩ) containing 3M NaCl placed at the stratum pyramidale/stratum oriens border. Responses were evoked by stimulating two electrodes placed within the Schaffer-collateral-commissural fibers. Baseline stimulation was every 150 seconds for each input with a 75 second interval between alternating inputs. Independence of inputs was assessed using a paired-pulse protocol. Stimulation intensity was adjusted such that the baseline population spike amplitude was approximately 40% of the maximum amplitude. Recordings were filtered at 3–10 kHz using an Axoclamp 2A Amplifier (Molecular Devices), and collected for online analysis at a sampling rate of 20kHz (Nguyen, 2013; Redondo et al., 2010) using WinLTP software (www.winltp.com; (Anderson and Collingridge, 2007).

2.3. Virus production

AAV-Sema4D-FL and AAV-CD4-FL were constructed by cloning the mouse Sema4D (NP_001268809.1 aa51 – aa861) or human CD4 (M12807.1 aa26-458) open reading frames (ORFs) into the AAV9.hsyn.eGFP plasmid (Addgene #105539) in place of GFP. Both constructs contained an artificial membrane-targeting signal sequence as well as a myc epitope tag upstream of the Sema4D and CD4 coding sequences. AAV-Sema4D-ECD and AAV-CD4-ECD constructs comprised the same N-terminal signal sequence and myc tag upstream of Sema4D NP_001268809.1 aa51 – aa733 and CD4 M12807.1 aa26-396 respectively. These ORFs were also cloned in place of GFP in the AAV9.hsyn1.GFP plasmid. In both CD4 constructs, an Asn-Ile mutation was inadvertently introduced at aa64. Following cloning, plasmids were packaged into AAV9 capsid and active virus was harvested and purified for use in vivo. Cloning and viral packaging and purification were performed by Vector Biolabs (Malvern, PA).

2.4. Organotypic slice culture

C57BL/6 mouse brains were dissected from P6-P8 animals of both sexes into cutting solution (126mM NaCl, 25mM NaHCO3, 3mM KCl, 1mM NaH2PO4, 20mM dextrose, 2mM CaCl2, 2mM MgCl2 in deionized water at 315-319 mOsm) (Stoppini et al., 1991). Coronal slices were taken at a thickness of 300 μm using a tissue chopper (Compresstome VF-200, Precisionary Instruments Inc.). Individual slices were placed on cell culture inserts (0.4 um pore size, Millipore). Organotypic culture media (2mM glutamax, 1mM CaCl2, 2mM MgSO4, 12.9mM d-glucose, 0.08% ascorbic acid, 18 mM NaHCO3, 35mM HEPES, 20% horse serum, 1 mg/mL insulin in minimum essential media) at pH 7.45 and 305 mOsm was added outside of the inserts. Slices were maintained for 6 days in vitro at 35°C and 5% CO2 with media replacements every other day.

One day after harvesting slices, a solution containing AAV9.hsyn viruses encoding either full-length or the ECD of either Sema4D or CD4 (produced and purified by Vector Biolabs) in combination with an equal amount of AAV9.hsyn.GFP virus (Addgene, #105539-AAV9) was pipetted onto the hippocampus within the slice (1 μL on each hemisphere, each virus at 2.55 x 1012 Gc/mL). A subset of slices were infected with only AAV9.hsyn.GFP and at DIV6 were treated with human Sema4D-Fc ectodomain/human Fc fusion protein (Sema4D-Fc; R&D Systems, #7470-S4) or Fc control protein (R&D Systems, #110-HG) at a final concentration of 2 nM/well for 2 hours before being fixed immediately following.

2.5. Immunofluorescence performed in organotypic slice cultures

To quantify synapse density at DIV6, organotypic slices were fixed in 4% paraformaldehyde/4% sucrose for 20 minutes at 4°C. After 3 x 10-minute washes in PBS, slices were incubated overnight in permeabilization solution (0.1% Triton-X in PBS) followed by an overnight incubation in blocking solution (20% bovine serum albumin with 0.1% Triton-X in PBS) at 4°C. Next, slices were incubated in anti-GAD65 (EMD Millipore, #MAB351) at 1:150 in blocking solution and incubated overnight at 4°C followed by 3 x 10-minute PBS washes and incubation with anti-mouse-Cy3 (Jackson Laboratories, #115-165-003) at 1:500 into secondary solution (blocking solution diluted 1:1000 in PBS) for 2 hours at room temperature. Following 3 x 10-minute PBS washes, slices were mounted (insert side down) on slides in Vectashield + DAPI mounting media (Vector Laboratories).

2.6. Imaging and analysis of organotypic slice cultures

16-bit images of neurons were acquired on a Zeiss LSM880 Confocal microscope using a PlanApochromat 63x/1.40 Oil DIC M27 objective. Within each experiment (slices isolated, infected, and processed for immunostaining in parallel on the same days), images were acquired with identical settings for laser power, detector gain, and amplifier offset. Settings were initially optimized across multiple control slices to avoid oversaturation of pixels. Images were acquired as z-stacks (5-15 optical sections, 0.5 μm step size) for each of 4-5 fields of view per hemisphere (134.95 μm x 134.95 μm) containing the pyramidal cell layer in CA1 from each slice. Within each z-stack, the image with the greatest fluorescent intensity for GAD65 signal was chosen for analysis; neurons were selected at random for analysis using only the GFP signal, thus blinding the experimenter to the GAD65 signal in the chosen neurons. Using the DAPI signal as a guide, the nuclei of these cells were traced and then a concentric perimeter expanding a uniform distance of 2 μm around the perimeter of the nucleus was drawn using the “draw band” function in ImageJ (NIH). This defines a doughnut-shaped region of interest (ROI) around the outer perimeter of the nucleus and provides a close approximation of the cell soma boundary encompassing the area where synapses form onto the cell membrane. Using ImageJ, the background signal was measured in three distinct places that lacked true synaptic signal in each image and the average mean background intensity was subtracted from the entire image. Finally, signal in the GAD65 channel was binarized, the adjustable watershed algorithm was applied, and GAD65 puncta within each ROI were quantified using the “analyze particles” function (size=0.1-10 and circularity=0.01-1.00). The number of puncta per ROI was divided by the ROI area to give a per soma density of GAD65 puncta. Between 16-40 cells were analyzed per slice. A subset of neurons in the CA1 region exhibit very high GAD65 immunofluorescence which fills the cell soma and are presumably interneurons. Such cells were excluded from analysis.

2.7. Animal surgeries for EEG headmount attachment, cannula placement and AAV-Sema4D-ECD virus injection

Male mice (10-12 weeks of age) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine and a prefabricated headmount (part # 8201; Pinnacle Technology, Inc) was affixed to the skull with four screws and dental cement. Two of the screws serve as differential EEG leads, which were placed two bilaterally anterior and two posterior to bregma, and the other screws serve as the reference ground and the animal ground. For the Sema4D infusion experiments, mice were implanted with a guide cannula into the dorsal hippocampus (A/P: −2.0mm; M/L: ± 1.5mm; D/V: −2mm) during EEG headmount attachment. The animals were allowed to recover for a minimum of 5 days before experimentation. For virally delivered Sema4D, 500nl of AAV-Sema4D (2.55 Gc/mL) or control virus was stereotaxically injected into the dorsal hippocampus during EEG headmount attachment 1 week prior to KA administration (20mg/kg, i.p.).

2.8. KA seizure paradigm

Sema4D (100nM, 500nl) or vehicle (0.9% sterile injection saline, 500nl) was infused into the hippocampus 1 hr prior to KA administration (20mg/kg, i.p.). For both Sema4D infused animals and virus injected animals, we administered 5mg/kg diazepam 2hrs after KA administration as previously described by our laboratory (Sivakumaran and Maguire, 2016).

2.9. Electroencephalogram (EEG) recording and analysis

EEG recordings from male C57BL/6 mice were carried out as previously described (Hooper et al., 2018; Sivakumaran and Maguire, 2016); recording and analyses were performed by two different experimenters with blinding to condition. Recordings were collected in awake, behaving animals using a 100x gain preamplifier high pass filtered at 1.0 Hz (Pinnacle Technology, part #8202-SE) and tethered turnkey system (Pinnacle Technology, part #8200).

The KA model of SE was employed for these studies because it is an acute, well-established model of TLE in which animals enter SE within an hour post-injection and develop seizures that do not respond to treatment with diazepam (Fritsch et al., 2010; Levesque and Avoli, 2013). Electrographic activity was recorded for 1hr before and 2hrs following KA administration. Diazepam was administered 2hr following KA administration and electrographic activity was recorded for an additional hour. In brief, epileptiform activity was considered to be paroxysmal activity having a sudden onset and an amplitude at least 2.5x the standard deviation of the baseline and a consistent change in the Power of the fast Fourier transform of the EEG. In the KA model, the animals typically enter SE by 1 hour post-KA administration. SE was defined as persistent, unremitting epileptiform activity lasting longer than 5 consecutive minutes. Our definition of “epileptiform activity” includes both discrete ictal events and periods of SE. These criteria have been used previously by our group (O'Toole et al., 2014,) (Maguire et al., 2005) as well as by other experts in the field (Castro et al., 2012). Seizure latency was defined as the time elapsed from the KA injection to the start of the first electrographic seizure. The % time epileptiform activity was calculated as the cumulative time of all epileptiform activity during a 120-min recording period divided by 120 min. The latency to SE was calculated as the time elapsed from the KA injection to the start of the SE (first 5 mins of unremitting epileptiform activity) while SE cessation was identified as elimination of epileptiform activity lasting for at least 5 minutes.

The diazepam sensitivity (as measured by a reduction in the power of the electrographic signal and cessation of epileptiform activity) was determined as previously described (Hooper et al., 2018; Sivakumaran and Maguire, 2016). Mice were considered to be diazepam-insensitive if diazepam treatment failed to suppress epileptiform activity within 10 mins of administration.

2.10. Immunohistochemistry and analysis for virus-injected animals:

Male C57BL/6 mice were injected with AAV-Sema4D-ECD or AAV-CD4-ECD virus as described above and sacrificed 2-3 weeks post-injection. The brains were removed and fixed in 4% paraformaldehyde for 24 hours, transferred to 10% sucrose for 24 hours, and then transferred to 30% sucrose for 24 hours. They were then embedded in Optimal Cutting Temperature (OCT) compound and frozen down for storage at −80°C. The brains were sectioned at 40 μm using a cryostat (Leica) and slices were suspended in PBS at 4°C until being processed for immunohistochemistry.

Representative slices from the hippocampus were chosen for staining. The sections were blocked in PBS with 10% normal goat serum (NGS) and 0.3% Triton for one hour at room temperature prior to incubation with the primary antibody mouse anti-GAD65 (EMD Millipore, MAB3551) at a concentration of 1:100 overnight at 4°C. The sections were then incubated in the secondary antibody (anti-Ms Alexa 488, Invitrogen, A32766) at 1:200 for two hours at room temperature and mounted with Vectashield Hardset antifade mounting medium with DAPI (Vector Laboratories, H-1500). Imaging was performed on a Leica SP8 Confocal microscope using a 40x oil objective. 2-3 images per hemisphere per section were taken of the hippocampus near A/P −2.0 (the virus injection site). Microscope and laser settings were kept constant across all images. Quantification of GAD65 immunofluorescence was performed using ImageJ. An outline was traced around the CA1 region of the hippocampus and the fluorescence intensity in the region of interest was quantified. For each image, the CA1 mean intensity was normalized to the mean intensity of background.

2.11. Statistical analysis

Statistical analyses were performed using the R programming language (R Core Team, 2022), Excel, or SPSS. For all experiments we assessed the basic properties of samples (Q-Q plots, residual plots, distributions etc.) prior to running statistical tests with distribution assumptions. Linear mixed effects modeling was conducted in R utilizing packages robustlmm and emmeans (Lenth, 2022). Bootstrapped multiple comparisons tests were conducted using custom written code in R. For counts data, we used the ‘N-1’ corrected form of the chi-squared test for the analysis of counts in one-margin fixed contingency tables (Campbell, 2007). Specific statistical tests are described in the text or figure legends.

3. Results

3.1. Application of the extracellular domain of Sema4D to acute hippocampal slices decreases excitability

Previously we observed that application of the ECD of Sema4D fused to the Fc region of human IgG (Sema4D-Fc) mediates an increase in GABAergic synapse density as evidenced by immunostaining and seizure suppression in rodent hippocampus (Acker et al., 2018). In addition, using whole cell voltage-clamp, we demonstrated that Sema4D-Fc application caused an increase in mini Inhibitory Postsynaptic Currents (mIPSC) in dissociated hippocampal neuronal cultures and acute slice, consistent with an increase in the density of functional GABAergic synapses (Kuzirian et al., 2013). Here, we sought to determine the effect and time course of Sema4D treatment on the hippocampal circuit. We utilized a recording configuration and set-up that allows the recording of stimulation-evoked field hippocampal activity for long periods of time (> 12 hours; (Redondo et al., 2010). Recordings were performed in the CA1 region of acute hippocampal slices obtained from adult male and female rats (aged postnatal day (P) 140-180). Rats were utilized because hippocampal slices obtained from rats are more robust for exceptionally long time-course electrophysiological recordings than are those obtained from mice. Evoked field responses comprise a positive-going field EPSP on which a negative-going population spike is superimposed. Following a stable 1-hour baseline, we bath-applied Sema4D-Fc or Fc control protein for 2 hours. A decrease in population spike amplitude was observed starting at ~1 hour after Sema4D-Fc perfusion began and lasted throughout the recording period, including for 3 hours post-washout of Sema4D-Fc (Figure 1A). We quantified this effect at 3 hours post-washout and found a significant Sema4D-Fc-mediated decrease in population spike amplitude (Figure 1Bi & Ci; alpha < 0.05 by bootstrapped multiple comparisons test with Holm-Šidák correction).

Figure 1. The extracellular domain of Sema4D protein decreases population spike amplitude in acute hippocampal slices.

Figure 1.

Open in a new tab

Sema4D-Fc treatment depressed population spike amplitude by ~50% while having no effect on fEPSP slope or paired-pulse facilitation (PPF) ratio. Acute hippocampal slices were isolated from rats of both sexes at 20-25 weeks old. Responses were recorded by low-frequency stimulation of the Schaffer-collateral commissural pathway by stimulating electrodes placed in the stratum radiatum and recording responses at the CA1 pyramidal/stratum oriens border. A stable baseline (1 hour) was obtained before drug application for 2hrs. Comparisons were made 3hrs post washout (i.e., at t=6hrs). A, Time plots represent the pooled effects (both n=8) of control Fc. (black) vs Sema4D-Fc (blue) application to evoked population spikes (Ai), fEPSP slope (Aii) and PPF (measured as the ratio of fEPSP slopes to 2 successive pulses delivered at a 50ms interval; Aiii). Shaded areas are the (bootstrapped) s.e.m. Traces above the time plots represent the raw data comprising averages of time points within each experiment taken at the time points indicated. Fc / Sema4D-Fc application are shown in light gray. For both conditions, vertical and horizontal scale lines indicate 1mV and 5ms, respectively. B, Raw values (points) and summary statistics (mean (bars) and SEM (lines)) of population spike amplitude (Bi), fEPSP slope (Bii) and PPF ratio (Biii) shown at baseline and test time points as in A for control Fc (left, black) vs Sema4D-Fc (right, blue). In the group receiving Sema4D-Fc treatment, the mean spike amplitude at test was significantly lower than its baseline (bootstrapped multiple comparisons test with Holm-Šidák correction); no other conditions differed significantly from one another; * indicates alpha < 0.05. C, Box plot to illustrate Cohen’s d (bootstrap) for population spike amplitude (Ci), fEPSP slope (Cii) and PPF ratio (Ciii). Mean and median effect sizes are represented by point and line, respectively. The interquartile range and 95% confidence interval are illustrated by box and whiskers, respectively. Significant effects from raw data (B) are also illustrated on this plot by asterisk for convenience.

In addition, we failed to observe a change in initial slope of the fEPSP (Figures 1Aii-Cii), a measure of L-glutamate mediated excitatory synaptic input, in the Sema4D-Fc treatment condition. We also did not observe a change in paired-pulse facilitation (Figures 1Aiii-Ciii), a measure of presynaptic release dynamics, in the Sema4D-Fc treatment condition. These data suggest that the depression of evoked firing observed upon Sema4D-Fc treatment is not due to a presynaptic effect which would dampen down excitatory synaptic transmission. Instead, given our previous studies demonstrating a rapid increase in GABAergic synapse formation in response to Sema4D application (Acker et al., 2018; Kuzirian et al., 2013), the most parsimonious explanation for the observed decrease in population spike amplitude is a Sema4D-dependent increase in evoked feedforward inhibitory transmission in the hippocampal slice.

3.2. Intrahippocampal infusion of the extracellular domain of Sema4D suppresses epileptiform activity and progression to SE in vivo

We reasoned that if a Sema4D-mediated decrease in evoked spiking activity in hippocampus is the result of the formation/stabilization of new GABAergic synapses, then infusion of Sema4D-Fc into the hippocampus should suppress seizure activity and restore BZD sensitivity in pharmacoresistant epilepsy. We chose the KA model of SE to test this hypothesis because it is an acute, well-established model of TLE in which animals enter SE within an hour post-injection and develop seizures that do not respond to treatment with diazepam (Fritsch et al., 2010; Levesque and Avoli, 2013). Furthermore, diazepam-insensitivity increases with prolonged seizure activity, which mimics the manifestation of clinical pharmacoresistance (Howard P. Goodkin, 2003).

We inserted a cannula unilaterally into the CA1 region of hippocampus in 10-12 week-old C57BL/6 male mice in addition to hippocampal depth electrodes for EEG recordings (see Methods). We infused 500nl of 100nM Sema4D-Fc or vehicle via cannula over five minutes, waited one hour, and then induced seizures by a 20mg/kg intraperitoneal (i.p.) injection of KA (Figure 2A). Seizure activity was monitored throughout the experiment using EEG. Epileptiform activity was defined as paroxysmal activity having a sudden onset and an amplitude at least 2.5x the standard deviation of the baseline and a consistent change in the Power of the fast Fourier transform of the EEG (Figures 2B-C). In addition, SE was defined as persistent, unremitting epileptiform activity lasting longer than 5 consecutive minutes (Hooper et al., 2018; Sivakumaran and Maguire, 2016). All animals achieved SE by 1 hour post KA injection.

Figure 2. Sema4D treatment suppresses seizures and progression to SE in vivo.

Figure 2.

Open in a new tab

A, Experimental timeline B, representative electrographic seizure activity from vehicle (black) and Sema4D-Fc-treated mice (red) and C, representative EEG power spectra from vehicle (top) and Sema4D-Fc treated mice (bottom). C57BL/6 male mice aged 10-12 weeks were used for this experiment. Sema4D-Fc was infused unilaterally into the CA1 region of the hippocampus 1hr prior to administration of kainic acid (20mg/kg, i.p.). Electrographic seizure activity was recorded for the entire 4hr period of the experiment. For the two hours following KA injection, the following characteristics were quantified: D, latency to onset of the first seizure; E, cumulative epileptiform activity (% time); F, number of mice that died; G, latency to SE; H, latency to cessation of SE; I, percent of diazepam insensitive mice which was quantified from the one hour following diazepam injection (5mg/kg i.p.). n = 8 mice per experimental group; unpaired Student’s t-test was performed on data in panels D,E,G,H; * denotes p<0.05 unpaired Student’s t-test. N-1 corrected chi-squared test was performed on data in panels F,I; no significant difference was found.

Analysis of the 2-hour recording period before diazepam treatment indicated that Sema4D-Fc infusion increased the latency to onset of the first ictal event (Figure 2D). In addition, Sema4D infusion decreased the percentage of time the animals spent exhibiting epileptiform activity compared to control animals (Figure 2E), quantified as the cumulative time of all epileptiform activity during a 120-min recording period divided by 120 min. As is typical in SE experiments, a number of animals did not survive SE (Figure 2F); a N-1 corrected chi-squared test failed to reveal a difference in mortality between conditions (p=.54). Nonetheless we did observe an increased latency to SE in the Sema4D-Fc treated group compared to control (Figure 2G) but no difference in latency to SE cessation (Figure 2H).

Two hours after KA injection, we delivered diazepam (5mg/kg) via i.p. injection and monitored the mice for one hour. We defined diazepam sensitivity as a reduction in the power of the electrographic signal and cessation of epileptiform activity as previously described (Hooper et al., 2018; Sivakumaran and Maguire, 2016). Mice were considered to be BZD-insensitive if diazepam treatment did not suppress SE within 10 mins of administration. By analyzing the EEG recordings obtained for 1 hour post-diazepam injection we observed that 2/6 animals in the Sema4D treatment group were insensitive to diazepam treatment compared to 6/7 animals in the control group (N-1 corrected chi-squared test p=.13) (Figure 2I). Taken together, these data indicate that hippocampal infusion of soluble Sema4D-Fc delays onset of KA-induced seizure, decreases epileptiform activity and delays progression to SE.

3.3. Adeno Associated Virus expressing Sema4D extracellular domain drives formation of inhibitory boutons in vitro

Delivery of Sema4D-Fc by intrahippocampal infusion via a surgically implanted cannula is cumbersome and only allows for acute delivery of Sema4D. To circumvent these issues, we developed virus-mediated delivery of the Sema4D ECD that would allow for both chronic and efficient delivery of the Sema4D protein.

We created Adeno-Associated Viruses (AAV) serotype 9 expressing either the full-length Sema4D protein (AAV-Sema4D-FL) or its ECD (AAV-Sema4D-ECD) and a control virus encoding full-length CD4 (AAV-CD4-FL), a transmembrane protein that has no effect on GABAergic synapse development (Raissi et al., 2013), or its ECD (AAV-CD4-ECD) (Figure 3A). To selectively drive expression in neurons, all constructs are expressed under control of the human Synapsin I promoter.

Figure 3. Validation of Sema4D-expressing adeno associated viruses in vitro.

Figure 3.

Open in a new tab

A, Schematic representing full-length or extracellular domain of Sema4D or CD4 protein encoded by the AAV viruses. B, Representative images of the CA1 principal cell layer in organotypic slices that were treated with Sema4D-Fc or Fc control proteins or infected with indicated AAV constructs; Sections are stained with an antibody that specifically recognizes GAD65 (red) and DAPI (blue). Scale bar represents 25μm. C, Density of GAD65 puncta per CA1 pyramidal cell soma; error bars are SEM. n = 233-351 neurons per treatment condition (represented by individual points), 16-40 neurons per slice, slices from 6-9 mice per treatment condition, 29 mice total. D, Box plot to illustrate the effect size, Cohen’s d. Mean effect size is represented by single points, interquartile range (IQR) and 95% confidence interval (CI) are illustrated by box and whiskers, respectively for the following comparisons: Fc vs Sema4D-Fc; AAV-CD4-FL vs AAV-Sema4D-FL; AAV-CD4-ECD vs AAV-Sema4D-ECD. A robust mixed effect linear model was fitted to the data with treatment as the fixed effect and animal and slice as random effects in order to control for variability that arises within animals and problems associated with pseudoreplication within slices (modeled as random intercepts) (Lazic et al., 2020). Consequently, data is presented as the estimated marginal means obtained from the model. Effect sizes (Cohen’s d), estimates of s.e.m, IQR, CI and Tukey-corrected multiple comparisons tests are based on these estimates * denotes p<0.05. Significant effects from raw data in C are also indicated by an asterisk in D for convenience.

As an initial test of efficacy of these viruses, we examined their ability to increase GABAergic synapse density in coronal, organotypic brain slices containing anterior hippocampus obtained from C57BL/6 mouse pups ages P6-P8. This time point was chosen for our initial verification because it is consistent with ours and others’ studies showing a pro-synaptogenic effect of purified Sema4D protein in ex vivo preparations (Acker et al., 2018; Frias et al., 2019; Kuzirian et al., 2013; McDermott et al., 2018; Raissi et al., 2013). After 1 day in vitro (DIV1), slices were infected with a combination of AAV9.hsyn.GFP virus (Addgene) to express GFP plus one of the Sema4D- or CD4-expressing viruses. At DIV6, slices were fixed and immunostained with an antibody that detects GAD65, an enzyme localized to the GABAergic presynaptic bouton (Figure 3B). As a positive control for the function of Sema4D, we also infected a subset of slices with only AAV9.hsyn.GFP at DIV1 and treated with either Sema4D-Fc or Fc recombinant protein for two hours at DIV6 before processing all slices for immunostaining (Figure 3B).

We determined GABAergic synapse density by imaging the pyramidal layer in the CA1 region of hippocampus and quantifying the density of GAD65-positive boutons formed onto the cell soma of GFP+ neurons similar to our previous study (Acker et al., 2018). We found that neurons infected with AAV-Sema4D-ECD exhibited a significantly higher density of GAD65 puncta per soma as compared to the AAV-CD4-ECD control (Figure 3C, D). Notably, infection with AAV-Sema4D-FL did not increase the density of GAD65 puncta. Our previous study showed that while the ECD of membrane-bound Sema4D is proteolytically cleaved, this cleavage is not required in order to promote GABAergic synapse development in dissociated neuronal cultures (Raissi et al., 2013). However, perhaps soluble Sema4D-ECD is a more efficacious signaling moiety than membrane bound Sema4D in the context of an ex vivo slice preparation. Importantly, the magnitude of change in GABAergic puncta density of ~20-25% observed in this experiment is comparable to those observed using purified Sema4D-Fc protein in both this experiment (Figure 3C, D, Fc vs. Sema4D-Fc) and our previous experiments (Acker et al., 2018). Based on these results, we conclude that chronic, viral delivery of Sema4D-ECD promotes GABAergic synapse formation similarly to treatment with the purified protein.

3.4. Delivery of AAV-Sema4D-ECD reduces epileptiform activity and delays progression to SE

We next elected to test AAV-Sema4D-ECD in the KA model of SE, to determine whether chronic viral overexpression of Sema4D-ECD could reduce seizure symptoms and impede progression to SE, similarly to infusion of purified Sema4D-Fc protein (Figure 2). To accomplish this goal, we performed bilateral injections of either AAV-Sema4D-ECD or control AAV-CD4-ECD virus into the CA1 region of hippocampus into 10-12 week-old C57BL/6 male mice; we also affixed headmounts for EEG recording. Following a 1-week recovery period, we performed a 20mg/kg i.p. injection of KA to induce seizures and monitored seizure activity using EEG recording as described above (Figures. 4A-C). Analysis of the two-hour recording period after KA administration and before diazepam treatment indicated no difference between AAV-CD4-ECD and AAV-Sema4D-ECD virus in latency to onset of the first seizure (Figure 4D), which differs from our results with infusion of purified Sema4D-Fc protein (Figure 2D), and perhaps reflects differences in the levels of Sema4D-ECD at the time of seizure induction due to different delivery methods. However, we did observe that administration of AAV-Sema4D-ECD significantly reduced the amount of time spent in epileptiform activity as compared to mice treated with the AAV-CD4-ECD virus (Figure 4E), similar to the effect observed with Sema4D-Fc infusion (Figure 2E), and consistent with an increase in inhibition in the hippocampi of these animals.

Figure 4. Delivery of AAV-Sema4D-ECD to hippocampus suppresses seizures and decreases severity of SE.

Figure 4.

Open in a new tab

A, Experimental timeline B, representative electrographic seizure activity from control AAV-CD4-ECD (black) and AAV-Sema4D-ECD treated mice (red) and C, representative EEG power spectra from control AAV-CD4-ECD (top) and AAV-Sema4D-ECD treated mice (bottom). C57BL/6 male mice aged 10-12 weeks were used for this experiment. AAV-Sema4D-ECD or control virus was injected into the DG region of the hippocampus 1wk prior to administration of KA (20mg/kg, i.p.). Electrographic activity was recorded for 1 hr prior to and 3hrs after KA injection which includes the 1 hour post diazepam administration (5mg/kg, i.p.). For the two hours following KA injection, the following characteristics were quantified: D, latency to onset of the first seizure; E, cumulative epileptiform activity (% time); F, number of mice that died (short red bar indicates 0 for AAV-Sema4D-ECD); G, latency to SE; H, latency to cessation of SE; I, percent of diazepam insensitive mice quantified from the one hour following diazepam injection (5mg/kg i.p.). n = 6-8 mice per experimental group; unpaired Student’s t-test was performed on data in panels D,E,G,H; N-1 corrected chi-squared test was performed on data in panels F,I. * denotes p<0.05 using a Student’s t-test; the chi squared test results were not significant.

Interestingly, in this experiment only 5/8 animals in the AAV-Sema4D-ECD group achieved SE by 2 hours post KA (all 8 animals were subsequently subjected to diazepam administration; see below), while 8/8 animals in the AAV-CD4-ECD cohort achieved SE by 1 hour post KA injection. Further, 2/8 control animals died during SE compared to 0/8 for the AAV-Sema4D-ECD group, no significant difference in mortality was observed between conditions (N-1 corrected chi-squared test p=.14) (Figure 4F). In addition, progression to SE was delayed in the group treated with AAV-Sema4D-ECD (Figure 4G) while there was no difference in latency to seizure cessation compared to the control group (Figure 4H), demonstrating a decrease in the duration of SE with Sema4D treatment. Taken together, these data suggest a meaningful ability of chronic Sema4D expression to decrease epileptiform activity and hinder progression to SE.Further, we speculate that the differences in severity of seizures post KA injection may be due to chronic expression of Sema4D in these animals, as opposed to the acute Sema4D-Fc treatment described above.

Two hours after KA injection, we delivered diazepam (5mg/kg) via i.p. and monitored the mice by EEG for an additional hour. Analysis of these recordings showed that 1/8 animals in the AAV-Sema4D-ECD treatment group were insensitive to diazepam treatment compared to 3/6 animals in the control group (N-1 corrected chi-squared test p=.14) (Figure 4I). To validate the targeting of our viral injections to hippocampus, we performed post hoc immunostaining for GAD65 on animals that had been injected with AAV-Sema4D-ECD or AAV-CD4-ECD (Figure S1). We found an ~20% increase in GAD65 immunostaining fluorescence intensity in the hippocampus of animals injected with AAV-Sema4D-ECD, consistent with correct targeting of the virus. Taken together, these results demonstrate that virally-mediated overexpression of Sema4D-ECD suppresses seizure activity, consistent with increased inhibition in the hippocampus.

4. Discussion

The present study advances the case for Sema4D-dependent GABAergic synapse formation as a new avenue for developing anti-seizure therapeutics based on the following observations. First, we demonstrated that evoked CA1 excitability is suppressed by application of Sema4D protein to acute hippocampal slices and that this effect endures for at least 3 hours, suggesting that Sema4D-mediated increased inhibition underlies the seizure suppression that we observe with in vivo application of Sema4D. Second, we showed that acute application of Sema4D protein in vivo decreases seizure susceptibility and delays seizure progression in the KA model of SE, consistent with a model whereby Sema4D promotes the formation and/or stabilization of GABAergic synapses. Third, we successfully developed a chronic, less invasive method to deliver Sema4D ECD using virus. We demonstrated that chronic delivery of Sema4D via viral transduction promotes increased GABAergic synapse density in ex vivo hippocampal organotypic slice cultures similar to purified Sema4D-Fc protein. Further, using the same KA model of SE, we discovered that chronic expression of the Sema4D-ECD in hippocampus in vivo decreases epileptiform activity and mitigates the progression to SE.

Taken together, these findings lead us to propose a model whereby Sema4D treatment promotes GABAergic synapse formation within 1 hour, resulting in a slower progression to seizure onset and SE, suppression of hippocampal network excitability, and resilience to hyperactivity, presumably via incorporation of new GABAergic synapses into the hippocampus. Importantly, these data support the therapeutic potential of AAV-Sema4D-ECD in seizure disorders.

Studies in multiple model systems including from our lab have demonstrated that Semaphorins and their receptors are critical mediators of synaptogenesis (Ding et al., 2011; Duan et al., 2014; Joo et al., 2013; Mizumoto and Shen, 2013; O'Connor et al., 2009; Tran et al., 2009; Uesaka et al., 2014). Sema4D regulates GABAergic synapse formation via signaling through its high affinity receptor Plexin-B1 (Kuzirian et al., 2013; McDermott et al., 2018). It is widely accepted that Sema4D binding to Plexin-B1 causes dimerization and activation of the PlexinB1 intracellular GTPase activating protein (GAP) domain, triggering downstream signal transduction events that regulate the actin cytoskeleton and cell morphology (Li et al., 2021; Negishi et al., 2005; Oinuma et al., 2004, 2006; Worzfeld et al., 2014).

Our current model of Sema4D-dependent synaptogenesis posits that Sema4D/Plexin-B1 signaling modulates the protein scaffolding machinery present at the synapse. Specifically, live-imaging of fluorescently-labeled synaptic proteins revealed that Sema4D signaling changes the subcellular localization of the GABAergic scaffolding protein gephyrin at the postsynapse (Kuzirian et al., 2013; Negishi et al., 2005). Rapid “splitting” of gephyrin-GFP puncta within ten minutes of Sema4D application resulted in additional gephyrin puncta which may nucleate new sites for GABAergic synapses to form. Furthermore, the c-Met receptor tyrosine kinase, which regulates actin depolymerization downstream of Sema4D/Plexin-B1 signaling (Swiercz et al., 2008), has been implicated in Sema4D-mediated stabilization of presynaptic GABAergic boutons and is specifically required in the presynaptic neuron for this effect (Frias et al., 2019). However, the precise signal transduction mechanisms underlying Sema4D/Plexin-B1 mediated GABAergic synaptogenesis are yet to be determined.

Both Sema4D and Plexin-B1 are expressed in inhibitory and excitatory neurons and glia in developing hippocampus (McDermott et al., 2018). Our functional studies show that Sema4D signals through Plexin-B1 expressed both pre- and postsynaptically, suggesting that a bidirectional trans-synaptic signaling complex may regulate Sema4D-dependent GABAergic synapse formation. Since Sema4D-ECD expression is driven by a neuron-specific promoter in the AAV vector, our results demonstrate that overexpression of Sema4D-ECD in neurons is sufficient to increase the density of functional inhibitory synapses in hippocampus (Figure 4). We favor a model whereby virally-delivered Sema4D-ECD, secreted from neurons, acts in both an autocrine and paracrine manner to regulate this process.

BZDs combat seizures and enhance inhibition by direct binding to GABAARs (Sills and Rogawski, 2020). However multiple groups showed that prolonged seizures are correlated with internalization or altered trafficking of GABAARs, with some studies citing decreased surface expression of the γ2 and β2/3 subunits of the GABAAR (Deeb et al., 2012; Goodkin et al., 2008; Goodkin et al., 2005; Naylor, 2005; Terunuma et al., 2008). Thus, therapeutics to combat SE that focus on methods to increase the density of GABAARs at the synapse to provide new sites for BZD binding could be effective as an add-on medication to BZD in pharmacoresistant SE. Future studies will be required to determine whether Sema4D alters GABAA receptor trafficking or localization.

The effect of ion flow through GABAARs can depolarize or hyperpolarize the neuron; this role changes throughout development and is mediated in large part by the constitutively active KCC2 K+-Cl− co-transporter and Cl− homeostasis (Deeb et al., 2012). Thus, an alternative hypothesis regarding BZD resistance asserts that changes in KCC2 function interfere with BZD efficacy by altering the effect of net Cl− flow across the cellular membrane, reversing the electrochemical driving force and the direction of GABAA-mediated current (Deeb et al., 2012; Moore et al., 2017).

Interestingly, a recent study reported a proteomics approach which identified KCC2-interacting proteins. This study showed that gephyrin interacts with KCC2 and regulates its function by promoting KCC2 clustering at the membrane (Al Awabdh et al., 2022). Given this finding, it is possible that another mechanism by which Sema4D restores BZD sensitivity is via gephyrin splitting (Kuzirian et al., 2013) which could cause a redistribution of gephyrin and therefore KCC2 at the membrane. Future studies will be required to determine whether Sema4D alters GABAA receptor and/or KCC2 trafficking or localization. Intriguingly the same proteomics study that identified the KCC2-gephyrin interaction also identified the Sema4D receptor Plexin-B1 as a putative interacting protein with KCC2 (Al Awabdh et al., 2022).

Taken together, our data provide proof of concept that Sema4D-mediated GABAergic synapse development could ameliorate the symptoms of intractable forms of epilepsy such as SE. We also provide evidence that viral-mediated transduction of Sema4D is an efficacious and promising delivery method. Irrespective of delivery route or mechanism of action, our current data support further investigation of the translatability of Sema4D treatment for seizure disorders.

5. Conclusion

The number of patients suffering from pharmacoresistant epilepsy is expected to increase with the globally aging population. Alternative therapies to treat this disorder include surgical resection, deep-brain stimulation, and targeted laser ablation (Rai and Drislane, 2018). All have disadvantages: surgeries/ablation are destructive and invasive; open-loop stimulations are only palliative seizure-reducing therapies. Sema4D could be introduced by microinjection of protein for acute management of seizures or as a gene therapy capable of reducing epileptiform activity and delaying onset of SE. The available evidence in rodent models of epilepsy suggests that, as a therapeutic, Sema4D has the potential to succeed where current anti-seizure medications fail.

Supplementary Material

1

Highlights:

  • Sema4D reduces epileptiform activity in the kainic acid model of status epilepticus

  • Sema4D reduces population spike amplitude in acute hippocampal slices

  • Chronically overexpressed Sema4D-ECD promotes GABAergic synapse formation

  • Virally delivered Sema4D reduces epileptiform activity and delays progression to SE

ACKNOWLEDGEMENTS:

We thank all members of the Paradis and Maguire labs for feedback and insight throughout the project.

FUNDING:

This work was supported by NIH grants R01 NS065856 (S.P.), F31 NS118799 (S.S.A.), R01 NS105628 and R01 NS102937 (J.M.).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ETHICAL PUBLICATION STATEMENT:

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

COMPETING INTERESTS:

Author Suzanne Paradis holds US Patent US-10626163-B2 entitled "Methods of Modulating GABAergic Inhibitory Synapse Formation and Function Using Sema4D." Co-inventors: Kuzirian, Marissa; Moore, Anna; Paradis, Suzanne. Author Suzanne Paradis is also co-founder and President of Severin Therapeutics, Inc. Author Jamie Maguire is on the Scientific Advisory Board for SAGE Therapeutics. The remaining authors have no conflicts of interest to disclose.

REFERENCES

  1. Acker DWM, Wong I, Kang M, Paradis S, 2018. Semaphorin 4D promotes inhibitory synapse formation and suppresses seizures in vivo. Epilepsia 59, 1257–1268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Al Awabdh S, Donneger F, Goutierre M, Seveno M, Vigy O, Weinzettl P, Russeau M, Moutkine I, Levi S, Marin P, Poncer JC, 2022. Gephyrin Interacts with the K-Cl Cotransporter KCC2 to Regulate Its Surface Expression and Function in Cortical Neurons. J Neurosci 42, 166–182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson WW, Collingridge GL, 2007. Capabilities of the WinLTP data acquisition program extending beyond basic LTP experimental functions. J Neurosci Methods 162, 346–356. [DOI] [PubMed] [Google Scholar]
  4. Campbell I, 2007. Chi-squared and Fisher-lrwin tests of two-by-two tables with small sample recommendations. Statistics in Medicine 26, 3661–3675. [DOI] [PubMed] [Google Scholar]
  5. Castro OW, Santos VR, Pun RY, McKlveen JM, Batie M, Holland KD, Gardner M, Garcia-Cairasco N, Herman JP, Danzer SC, 2012. Impact of corticosterone treatment on spontaneous seizure frequency and epileptiform activity in mice with chronic epilepsy. PLoS One 7, e46044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cruickshank M, Imamura M, Counsell C, Aucott L, Manson P, Booth C, Scotland G, Brazzelli M, 2022. Management of the first stage of convulsive status epilepticus in adults: a systematic review of current randomised evidence. Journal of Neurology. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deeb TZ, Maguire J, Moss SJ, 2012. Possible alterations in GABAA receptor signaling that underlie benzodiazepine-resistant seizures. Epilepsia 53 Suppl 9, 79–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ding JB, Oh WJ, Sabatini BL, Gu C, 2011. Semaphorin 3E-Plexin-D1 signaling controls pathway-specific synapse formation in the striatum. Nat Neurosci 15, 215–223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duan Y, Wang SH, Song J, Mironova Y, Ming GL, Kolodkin AL, Giger RJ, 2014. Semaphorin 5A inhibits synaptogenesis in early postnatal- and adult-born hippocampal dentate granule cells. Elife 3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frias CP, Liang J, Bresser T, Scheefhals L, van Kesteren M, van Dorland R, Hu HY, Bodzeta A, van Bergen En Henegouwen PMP, Hoogenraad CC, Wierenga CJ, 2019. Semaphorin4D Induces Inhibitory Synapse Formation by Rapid Stabilization of Presynaptic Boutons via MET Coactivation. J Neurosci 39, 4221–4237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fritsch B, Stott JJ, Joelle Donofrio J, Rogawski MA, 2010. Treatment of early and late kainic acid-induced status epilepticus with the noncompetitive AMPA receptor antagonist GYKI 52466. Epilepsia 51, 108–117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goodkin HP, Joshi S, Mtchedlishvili Z, Brar J, Kapur J, 2008. Subunit-specific trafficking of GABA(A) receptors during status epilepticus. J Neurosci 28, 2527–2538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goodkin HP, Yeh JL, Kapur J, 2005. Status epilepticus increases the intracellular accumulation of GABAA receptors. J Neurosci 25, 5511–5520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hooper A, Fuller PM, Maguire J, 2018. Hippocampal corticotropin-releasing hormone neurons support recognition memory and modulate hippocampal excitability. PLoS One 13, e0191363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goodkin Howard P., M.D., Ph. D. and Kapur Jaideep, M.D., Ph.D., 2003. Responsiveness of Status Epilepticus to Treatment with Diazepam Decreases Rapidly as Seizure Duration Increases. Epilepsy Currents 3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Joo WJ, Sweeney LB, Liang L, Luo L, 2013. Linking cell fate, trajectory choice, and target selection: genetic analysis of Sema-2b in olfactory axon targeting. Neuron 78, 673–686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kuzirian MS, Moore AR, Staudenmaier EK, Friedel RH, Paradis S, 2013. The class 4 semaphorin Sema4D promotes the rapid assembly of GABAergic synapses in rodent hippocampus. J Neurosci 33, 8961–8973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lazic SE, Mellor JR, Ashby MC, Munafo MR, 2020. A Bayesian predictive approach for dealing with pseudoreplication. Scientific Reports 10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lenth RV, 2022. Estimated Marginal Means, aka Least-Squares Means, R package version 1.7.3. [Google Scholar]
  20. Levesque M, Avoli M, 2013. The kainic acid model of temporal lobe epilepsy. Neurosci Biobehav Rev 37, 2887–2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li ZL, Muller-Greven J, Kim S, Tamagnone L, Buck M, 2021. Plexin-Bs enhance their GAP activity with a novel activation switch loop generating a cooperative enzyme. Cell Mol Life Sci 78, 1101–1112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Madzar D, Geyer A, Knappe RU, Gollwitzer S, Kuramatsu JB, Gerner ST, Hamer HM, Huttner HB, 2016. Association of seizure duration and outcome in refractory status epilepticus. J Neurol 263, 485–491. [DOI] [PubMed] [Google Scholar]
  23. Maguire JL, Stell BM, Rafizadeh M, Mody I, 2005. Ovarian cycle-linked changes in GABA(A) receptors mediating tonic inhibition alter seizure susceptibility and anxiety. Nat Neurosci 8, 797–804. [DOI] [PubMed] [Google Scholar]
  24. McDermott JE, Goldblatt D, Paradis S, 2018. Class 4 Semaphorins and Plexin-B receptors regulate GABAergic and glutamatergic synapse development in the mammalian hippocampus. Mol Cell Neurosci 92, 50–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mayer SA, Claassen J, Lokin J, Mendelsohn F, Dennis LJ, Fitzsimmons BF Refractory status epilepticus: frequency, risk factors, and impact on outcome Arch. Neurol, 59 (2) (2002), pp. 205–210, 10.1001/archneur.59.2.205 [DOI] [PubMed] [Google Scholar]
  26. Mizumoto K, Shen K, 2013. Interaxonal interaction defines tiled presynaptic innervation in C. elegans. Neuron 77, 655–666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moore YE, Kelley MR, Brandon NJ, Deeb TZ, Moss SJ, 2017. Seizing Control of KCC2: A New Therapeutic Target for Epilepsy. Trends Neurosci 40, 555–571. [DOI] [PubMed] [Google Scholar]
  28. Naylor DE, 2005. Trafficking of GABAA Receptors, Loss of Inhibition, and a Mechanism for Pharmacoresistance in Status Epilepticus. Journal of Neuroscience 25, 7724–7733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Negishi M, Oinuma I, Katoh H, 2005. Plexins: axon guidance and signal transduction. Cell Mol Life Sci 62, 1363–1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nguyen P, 2013. Electrophysiological and Behavioral Approaches to the Analysis of Synaptic Tagging and Capture, in: Nguyen P (Ed.), Multidisciplinary Tools for Investigating Synaptic Plasticity Humana; Totowa, New Jersey, United States, pp. 179–195. [Google Scholar]
  31. O'Connor TP, Cockburn K, Wang W, Tapia L, Currie E, Bamji SX, 2009. Semaphorin 5B mediates synapse elimination in hippocampal neurons. Neural Dev 4, 18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Toole KK, Hooper A, Wakefield S, Maguire J, 2014. Seizure-induced disinhibition of the HPA axis increases seizure susceptibility. Epilepsy Res 108, 29–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Oinuma I, Katoh H, Negishi M, 2004. Molecular dissection of the semaphorin 4D receptor plexin-B1-stimulated R-Ras GTPase-activating protein activity and neurite remodeling in hippocampal neurons. J Neurosci 24, 11473–11480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Oinuma I, Katoh H, Negishi M, 2006. Semaphorin 4D/Plexin-B1-mediated R-Ras GAP activity inhibits cell migration by regulating beta(1) integrin activity. J Cell Biol 173, 601–613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. R Core Team, 2022. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. [Google Scholar]
  36. Rai S, Drislane FW, 2018. Treatment of Refractory and Super-refractory Status Epilepticus. Neurotherapeutics 15, 697–712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Raissi AJ, Staudenmaier EK, David S, Hu L, Paradis S, 2013. Sema4D localizes to synapses and regulates GABAergic synapse development as a membrane-bound molecule in the mammalian hippocampus. Mol Cell Neurosci 57, 23–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reddy DS, Kuruba R, 2013. Experimental models of status epilepticus and neuronal injury for evaluation of therapeutic interventions. Int J Mol Sci 14, 18284–18318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Redondo RL, Okuno H, Spooner PA, Frenguelli BG, Bito H, Morris RG, 2010. Synaptic tagging and capture: differential role of distinct calcium/calmodulin kinases in protein synthesis-dependent long-term potentiation. J Neurosci 30, 4981–4989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sills GJ, Rogawski MA, 2020. Mechanisms of action of currently used antiseizure drugs. Neuropharmacology 168, 107966. [DOI] [PubMed] [Google Scholar]
  41. Sivakumaran S, Maguire J, 2016. Bumetanide reduces seizure progression and the development of pharmacoresistant status epilepticus. Epilepsia 57, 222–232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stoppini L, Buchs PA, Muller D, 1991. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37, 173–182. [DOI] [PubMed] [Google Scholar]
  43. Swiercz JM, Worzfeld T, Offermanns S, 2008. ErbB-2 and met reciprocally regulate cellular signaling via plexin-B1. J Biol Chem 283, 1893–1901. [DOI] [PubMed] [Google Scholar]
  44. Terunuma M, Xu J, Vithlani M, Sieghart W, Kittler J, Pangalos M, Haydon PG, Coulter DA, Moss SJ, 2008. Deficits in phosphorylation of GABA(A) receptors by intimately associated protein kinase C activity underlie compromised synaptic inhibition during status epilepticus. J Neurosci 28, 376–384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Towne AR, Pellock JM, Ko D, DeLorenzo RJ, 1994. Determinants of mortality in status epilepticus. Epilepsia 35, 27–34. [DOI] [PubMed] [Google Scholar]
  46. Tran TS, Rubio ME, Clem RL, Johnson D, Case L, Tessier-Lavigne M, Huganir RL, Ginty DD, Kolodkin AL, 2009. Secreted semaphorins control spine distribution and morphogenesis in the postnatal CNS. Nature 462, 1065–1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Trinka E, Cock H, Hesdorffer D, Rossetti AO, Scheffer IE, Shinnar S, Shorvon S, Lowenstein DH, 2015. A definition and classification of status epilepticus - Report of the ILAE Task Force on Classification of Status Epilepticus. Epilepsia 56, 1515–1523. [DOI] [PubMed] [Google Scholar]
  48. Uesaka N, Uchigashima M, Mikuni T, Nakazawa T, Nakao H, Hirai H, Aiba A, Watanabe M, Kano M, 2014. Retrograde semaphorin signaling regulates synapse elimination in the developing mouse brain. Science 344, 1020–1023. [DOI] [PubMed] [Google Scholar]
  49. Worzfeld T, Swiercz JM, Senturk A, Genz B, Korostylev A, Deng S, Xia J, Hoshino M, Epstein JA, Chan AM, Vollmar B, Acker-Palmer A, Kuner R, Offermanns S, 2014. Genetic dissection of plexin signaling in vivo. Proc Natl Acad Sci U S A 111, 2194–2199. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1900984-supplement-1.pdf (155.9KB, pdf)

RESOURCES