Epilepsy is a neurological disorder characterized by recurrent seizures, often resulting from neural injury. However, all antiepileptic

medications prescribed today affect ion channel permeability as well as other targets with the ultimate goal of restoring the balance between

gamma-aminobutyric acid (GABA) and glutamate – the main inhibitory and excitatory neurotransmitters in the central nervous system (CNS),

respectively. Epileptogenesis, the process whereby a normal brain becomes epileptic, remains poorly understood and untreated. Recent studies

suggest that following neural injury, axonal sprouting and subsequent aberrant synapse formation is one mechanism that may facilitate the

development of epilepsy. The elucidation of the cellular and molecular mechanisms of epileptogenesis, then, may provide novel treatments aimed

at prophylaxis, as opposed to current therapies, which are exclusively symptomatic.

Temporal lobe epilepsy is the most common form of partial epilepsy in which seizures emerge from a focus. In humans, temporal

lobe epilepsy most often shows seizure focus in the hippocampus. Its cause, however, remains enigmatic. Nonetheless, several studies have shown

that epileptic seizures arising from the limbic system, which includes the hippocampus, are associated with cell death and synaptic reorganization of

hippocampal circuitry in both animal models and humans. One line of evidence that we follow here is the role that the secreted protein semaphorin

3F (Sema3F) may play as a guidance molecule.

During development, the nervous system follows a genetic program to establish and organize axon pathways as well as neural

circuitry via an array of axon guidance molecules. Several families of axon growth-promoting and growth-inhibiting extracellular proteins that

mediate the formation of neural circuitry in the developing brain have been identified, including the semaphorins (Fig. 1). The semaphorins are a

large family of guidance cues with roles in axon guidance, cell migration, and dendritic development in vivo. In particular, Class 3 semaphorins are

secreted proteins and are comprised of six family members, Sema3A-3F. They signal through receptor complexes that contain a ligand-binding

subunit and a signal transducing component, encoded by the neuropilin (Npn) and plexin gene families, respectively. Most notably, Sahay et al.

(2003) have shown that the protein Sema3F is required for the proper patterning of the limbic system circuitry, which includes the hippocampus.

More specifically, Sema3F knockout mice have defects in hippocampal circuitry, especially in projections of the infrapyramidal tract (IPT) and

main mossy fibers (MF), when compared to their wild-type littermates (Fig. 2).

Unlike their roles in neural development, a function for semaphorins in the adult brain is not known. In situ hybridization (ISH)

analysis performed by Barnes et al. (2003), though, indicates that secreted semaphorins are expressed in a variety of distinct cell types in the adult

hippocampus, including granule cells of the dentate gyrus (DG) and pyramidal neurons of the CA3 and CA1 layers of the hippocampus. Barnes et

al. (2003) also studied semaphorin gene expression in an adult rat model of synaptic reorganization, kainic acid (KA)-induced status epilepticus

(SE), in which KA treatment results in nearly continuous limbic seizures for several hours, leading to cell death of CA3 pyramidal cells,

synaptogenesis, and the development of epilepsy weeks to months later. In addition to being spatially restricted, secreted semaphorin expression

appears to be modulated by neural activity. Within the first week after KA-induced SE, semaphorin 3F mRNA content was significantly decreased

in CA1 and CA3 neurons. Moreover, the pathology observed is similar to temporal lobe epilepsy. Together, the above evidence suggests the

hypothesis that secreted semaphorins play a novel role in synaptic reorganization in the adult brain and thereby promote epileptogenesis. To test

this hypothesis, we focused on Sema3F and its receptor Npn-2 in the adult brain, analyzing electroencephalographic (EEG) recordings of mice

deficient in Sema3F-Npn-2 signaling. We also undertook depth electrode implantation and depth EEG recordings as a way to pinpoint the origin of

epileptic activity in these mice. Here, we introduce the possibility that the Sema3F knockout mouse model is a new model for temporal lobe

epilepsy.

Semaphorin 3F Knockout Mouse Model: A New Model for Temporal Lobe Epilepsy

Sophia F. Shakur, Teemu Tha, Michael S. Tsimis, John Chua-Tuan, and Jehuda P. SepkutyDepartment of Neurology, Johns Hopkins University, Baltimore, Maryland

Abstract

Introduction

Methods

Scalp EEG Surgery

Animals were anesthetized with 2,2,2-tribromoethanol (250 mg/kg). After shaving and cleansing the surgical site,

a 1 cm longitudinal incision was made on the scalp. Two screws (Small Parts Inc.) were attached to the skull, approximately 6

mm posterior to bregma and 5 mm on either side of the saggital suture. Another screw was attached2 mm anterior to bregma and

2 mm on one side of the saggital suture. A tripolar electrode unit (Plastics One Inc.) was cut, with each wire 1 cm long. The end

of each wire was looped around the scre. The entire electrode unit was secured on the head with cranioplastic cement.

Depth Electrode Implantation

Animals were anesthetized with 2,2,2-tribromoethanol (250 mg/kg). After shaving and cleansing the surgical site,

a 1 cm longitudinal incision was made on the scalp. Animals were then secured in a dual manipulator stereotaxic instrument

(Stoelting Company). Using the stereotaxic manipulator, a small burr hole was drilled in the skull over the frontal lobe, 2 mm

anterior to bregma and 3 mm lateral to the saggital suture, on each side of the brain. Another burr hole was drilled in the skull over

the right dorsal hippocampus, 1.8 mm posterior to bregma and 2.7 mm lateral to the saggital suture. A stainless steel

microelectrode (FHC Inc.) was then lowered into the hippocampus, 1.8 mm below the surface of the brain. The depth electrodes

were connected to a multi-channel electrode pedestal (Plastics One Inc.). The exposed skull, the electrodes, and the base of the

multi-channel electrode pedestal were finally covered with cranioplastic cement.

EEG Recordings

Following a 24-hour recovery period after surgery, animals were placed daily in the EEG recording chamber,

which allowed unrestrained movement while recording. Signals were recorded using Grass EEG machine model 8-16. EEG was

sampled for twenty minutes or until seizure activity occurred. If seizure activity occurred, observation was continued until five

minutes of non-epileptiform EEG activity had been recorded. Clinical behavior was monitored during the EEG recording. The

EEG was evaluated by counting spikes and by looking at spike discharges per minute staring. Recording and interpretation were

done separately and blinded.

Behavior Evaluation

Animals were observed daily during a one-hour non-invasive behavior test and during EEG recording. The

following were considered potential indicators of epileptic activity: staring (with or without vibrissa), jerking, rotatory

movements, vervisive head turns, tonic motor movements, and convulsions.

Pentylenetetrazol (PTZ) Test

To assess seizure susceptibility, animals were given an intraperitoneal (i.p.) injection of the epilepsy-inducing

agent PTZ (4mM at 100 mg/kg). EEG was recorded prior to PTZ administration to establish a baseline. EEG was also recorded

throughout the experiment. The first clinical sign of epilepsy, usually a twitch of the tail and/or sudden jerk of the body, was

accompanied by initial change in the EEG and was defined as an epileptic change. This behavior was followed by a generalized

convulsive seizure. Behavior, seconds to first twitch, seconds to generalized seizure, and duration of seizure were documented.

Results

Depth Electrodes Can Be Implanted Into the Hippocampus

Figure 7. Histological studies of depth electrode implantation.

Hematoxylin and eosin (H&E) staining of 8-10μm brain slices from wild-type mice show

that hippocampus is being targeted.

Figure 1. Long- and short-range axonal guidance cues. Class

3 semaphorins are critical for axon guidance, dendritic branching,

and cell migration during neural development. Many also

continue to be expressed in adult brain structures that retain a

high degree of synaptic plasticity, such as the hippocampus.

Figure 2. Sema3F-Npn-2 signaling is required for proper targeting of

infrapyramidal tract in CA3. A,B Without Sema3F (minus signs),

infrapyramidal tract (IPT, orange arrow) axons aberrantly extend into stratum

oriens (so) of CA3. C,D Calbindin immunostaining of brain slices with

abnormal IPT projection in sema3F null mice (D) versus wild-types (C).

Aside from its established role in neural development, the guidance molecule semaphorin 3F (Sema3F) appears to play a novel role in

synaptic reorganization in the adult brain. In particular, Sema3F continues to be expressed in a variety of cell types in the adult hippocampus and has

decreased expression in an adult rat model of synaptic reorganization, kainic acid-induced status epilepticus. It is thought that aberrant synapse

formation may contribute to epileptogenesis, the process whereby a normal brain becomes epileptic. The objective of this study was to investigate

the association between aberrant circuitry in Sema3F knockout mice and epileptogenesis. The study was conducted by implanting depth electrodes

into the hippocampus of Sema3F knockout mice and wild-type controls. Electroencephalograms (EEG) were recorded and behavior was monitored

daily. Scalp EEG recordings showed that Sema3F knockouts, in comparison to wild-type littermates, developed seizures. Depth EEG recordings

revealed that administration of pentylenetetrazol (PTZ; 100 mg/kg, i.p.) produced seizures in wild-types and Sema3F knockouts that first occurred in

the hippocampus and then spread to the scalp. Sema3F knockouts were also seen to have increased susceptibility to seizures compared to wild-types

with the same PTZ treatment. In humans, temporal lobe epilepsy most often shows seizure focus in the hippocampus. Alterations in hippocampal

circuitry underlie epileptogenesis in Sema3F knockout mice, providing a new model for temporal lobe epilepsy that can be exploited to develop

treatments to prevent epilepsy.

Acknowledgments

This work was supported in part by a Fall 2004 Provost’s Undergraduate Research Award (Hodson Trust, Johns Hopkins

University).

Sema3FKnockout Mice Develop Seizures

Figure 3. Scalp EEG recordings in Sema3F knockout mice. A typical EEG from a wild-type mouse (A) while stationary and

(C) while exploring. A typical EEG from a sema3F null littermate (B) while stationary and (D) while exploring. (B) This EEG

from a sema3F null reveals high voltage spikes and polyspike discharges for a duration of about 3-5 seconds. (E) In comparison

to wild-type littermates sema3F null mice showed more than a five-fold increase of spikes per minute of total recording. Wild-

type mice did not differ significantly from sema3F null mice in number of stationary episodes. n=6 (wild-type), n=8 (sema3F

nulls), (*p=0.0009) (two-tailed t-test).

Npn-2 Null Mice Show Epileptic Behavior

Figure 5. Npn-2 behavioral scoring. Wild-type mice differ significantly from npn-2-/-

null mice in exploratory episodes (*p=0.012) (two-tailed t-test) as well as in stationary

episodes (*p=0.005) (two-tailed t-test). n=7 (wild-type), n=4 (npn-2-/- nulls).

Sema3F Knockout Mice Have a Lower Seizure Threshold

PTZ-Induced Seizures Begin In the Hippocampus

Figure 9. EEG recordings of PTZ-induced seizures in wild-type and sema3F knockout mice

with depth electrodes implanted. RH, Right Hippocampal Depth Electrode; LF, Left Frontal

Screw; RF, Right Frontal Screw. Seizure Onset: Abnomal EEG activity (change in EEG from

baseline) first occurs in RH (depth). Seizure Propagation: Abnormal EEG activity later occurs in

LF and RF (scalp). Seizures recorded from wild-type and sema3F null start in the right

hippocampus (RH) and then propagate to the scalp over the frontal lobe (LF, RF) before

becoming generalized.

Figure 8. PTZ Test in Wild-Type versus Sema3F Knockout Mice. Sema3F null mice

have decreased latency for PTZ-induced seizures compared to wild-types with the same

PTZ treatment (100 mg/kg, i.p.).

Conclusions

Sema3F knockout mice have epileptic seizures manifested by staring and epileptiform EEG

during staring episodes.

Npn-2 null mice show epileptic behavior and there is early evidence indicating that npn-2-/-

nulls develop seizures.

Depth EEG recordings and PTZ tests indicate that Sema3F knockout mice are more susceptible

to seizures and that these seizures occur earlier in the hippocampus and then spread to the scalp.

These observations may be the result of developmental defects in Sema3F knockout mice or

may reflect a requirement for Sema3F-Npn-2 signaling in the adult brain.

Further investigation is needed to distinguish between these two possibilities.

This study introduces the possibility that the Sema3F knockout mouse is a new model for

temporal lobe epilepsy and, if proven to be due to synaptic modifications in the adult brain, may

lead to new treatments to prevent epilepsy.

Sema3F Knockout Mice Show Epileptic Behavior

Figure 4. Sema3F behavioral scoring. Wild-type mice differ from sema3F null mice

in exploratory episodes (p=0.0954) (two-tailed t-test) as well as in stationary episodes

(p=0.0633) (two-tailed t-test). n=3 (wild-type), n=3 (sema3F nulls).

Wild-type Null

Npn-2 Null Mice Show Seizure Activity

Figure 6. Scalp EEG recordings in Npn-2 null mice. Wild-type, an example of a typical

EEG from a wild-type mouse while exploring. A normal mixture of frequencies without

spikes was seen. Null, an example of a typical EEG from the npn-2-/- null mouse while

stationary reveals high voltage spikes and polyspike discharges (*). Null EEG also

correlates with epileptic behavior manifested in npn-2-/- null mice in the form of staring (st)

and jerking (j) episodes. I II

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References

Sahay A, Molliver ME, Ginty DD, Kolodkin AL (2003) Semaphorin 3F is critical for development of limbic system circuitry ad is

required in neurons for selective CNS axon guidance events. J Neurosci 23:6671-6680.

Sahay A, Kim C-H, Sepkuty JP, Cho E, Huganir RA, Ginty DD, Kolodkin AL (recently published in J Neurosci) Secreted

semaphorins modulate synaptic transmission in the adult hippocampus.

Barnes G, Puranam RS, Luo Y, McNamara JO (2003) Temporal specific patterns of semaphorin gene expression in rat brain after

kainic acid-induced status epilepticus. Hippocampus 13:1-20.

Tessier-Lavigne M, Goodman CS (1996) The molecular biology of axon guidance. Science 274:1123-1133.