Assessing post-synaptic partners of Dentate Granule Cells in a Model of Temporal Lobe Epilepsy

Grant A. Pizzo1

1Department of Neurology 5021 Biomedical Sciences Research Building Ann Arbor, MI 48109

Temporal lobe epilepsy (TLE) is the most common type of epilepsy among adults, affecting hundreds of thousands of Americans (Kwan & Bodie 2000; Sharma et al. 2007). In order to discover new treatments for the symptoms and prevention of the disease, pathology TLE must be understood at the cellular and molecular level. In both human patients and animal models, the dentate gyrus of the hippocampus is profoundly altered in this disease. Dentate granule cells (DGCs) in TLE tissue show abnormal axonal terminals, mossy fiber sprouting (MFS), and ectopic migration into the hilus (Kron et al 2010). Recent work indicates that these abnormalities are mostly limited to immature DGCs (Parent et al 2006; Parent et al 1997; Scharfman et al 2003). However, the overall influence of altered neurogenesis and DGC irregularities on epileptogenesis is unclear. In order to understand the role of DGC axon plasticity in TLE, we aim to determine how axon terminals and post-synaptic partners are changed in an epileptic environment. As part of an ongoing experiment, I induced SE using pilocarpine for animals postnatal day 56 (P56), injected retroviral reporters (RV) to label dividing DGCs at P60. 10 weeks after SE induction, tissues were perfused and immunohistochemistry was performed. To identify post-synaptic partners of DGCs, I have piloted three different antibodies used for immunostaining, two to identify interneurons, and one to identify mossy cells. Results thus far indicate that anti GAD67 and anti GluR2 antibodies were able to label the correct cells and will be refined for use in future experiments.

IntroductionTemporal lobe epilepsy (TLE) is often characterized by persistent seizures and memory dysfunction. Persons with epilepsy are significantly more likely to suffer from anxiety, personality disorder, cognitive impairment, and, most commonly, depression (Hoppe et al 2007; Perini et al 1996; Stefanello et al 2010). Despite extensive research, little is known about the cellular and molecular mechanisms causing a previously non-epileptic brain to begin generating spontaneous seizures. In both human and rodent models of TLE status epilepticus (SE) stimulates dentate granule cell (DGC) neurogenesis and is associated with extensive interneuron cell death in the hilus (Parent et al 1997; de Lanerolle et al., 1989) It is important to note that DGC neurogenesis normally occurs throughout adulthood, but is abnormally upregulated after SE. Cells that are born after SE are more likely to show morphological abnormalities that are believed to promote seizures (Kron et al. 2010). These abnormalities include DGC migration into the hilus, abnormal axon structure, and persistant hilar basal dendrites (HBD) (Parent et al 2006; Parent et al 1997; Scharfman et al 2003).

However, mature DGCs are largely resistant to alterations, suggesting epileptic insults influence mostly developing or newborn DGCs (Kron et al. 2010). One of the more intriguing abnormalities occurs when the axonal collaterals of DGCs aberrantly integrate into the inner molecular layer (IML). Normally, DGC axons

travel through the hilus and synapse with dendrites of interneurons in the hilus or pyramidal neurons of CA3. But in TLE, DGC axons may synapse with dendrites of other DGCs. This phenomenon, known as mossy fiber sprouting (MFS), is present in TLE tissue from humans and animal models and is proposed to have a role in epileptogenesis 1990; Sutula et al 1989; Tauck & Nadler 1985).

In addition to aiding my colleagues with parts of their projects, my role in the research was to test different antibodies for use in immunohistochemistry of tissues. More specifically, I piloted out several different antibodies to see which dilutions and antibody combinations labeled the correct cell types and at the same time having strong signals when viewed under an epifluorescence microscope.

The three antibodies I tested were anti GAD67, anti mGluR1/5 and anti GluR2. The findings indicate that anti GAD67and anti GluR2 were able to label the correct cell types and yield suitable images for epifluorescence microscopy. However, improvements need to be made with both antibodies for optimal results.

Materials and MethodsAnimals. All animal procedures were performed in accordance with protocols approved by the University Committee on Use and Care of Animals of the University of Michigan. We use male Sprague Dawley

rats. Animals were purchased from Charles River and kept under a constant 12 h light/dark cycle with access to food and water ad libitum.

Pilocarpine-induced SE. Adult male Sprague Dawley rats [postnatal day 56 (P56)] were pretreated with atropine methylbromide (5 mg/kg, i.p.); Sigma-Aldrich) and 20 min later were given pilocarpine hydrochloride (340 mg/kg, i.p.; Sigma-Aldrich) to induce SE. If convulsive seizure activity was not initiated within 1 h of the initial pilocarpine hydrochloride dose, an additional dose of 170 mg/kg was given. Seizures were monitored behaviorally and then terminated with diazepam (10 mg/kg) after 90 min of SE as previously described (Parent et al., 2002, 2006). Controls received the same treatments as experimental animals except that they were given saline in place of pilocarpine.

Intrahippocampal RV injections. At P60, animals were anesthetized with a ketamine/xylazine mixture and positioned in a Kopf stereotaxic frame. Then, a midline scalp incision was made, the scalp was reflected by hemostats to expose the skull, and bilateral burr holes were drilled. Synaptophysin-yellow fluorescent protein (Syp-YFP) RV vector (2.5 μl of viral stock solution/site) was injected into the left and right dentate gyri over 20 min each using a 5 μl Hamilton syringe, and the syringe needle was left in place for an additional 2 min before removing. Coordinates for injections (in millimeters from bregma suture and millimeters depth below the skull) caudal 3.9; lateral 2.3, depth 4.2.

Tissue processing and immunohistochemistry. 10 weeks after SE, animals were deeply anesthetized and perfused with 4% paraformaldehyde (PFA). The brains were removed, postfixed overnight in 4% PFA, cryoprotected in 30% sucrose, and frozen. Coronal sections (60 μm thick) were cut with a cryostat, and fluorescence immunohistochemistry performed on free-floating sections (Parent et al., 1997, 1999). Sections were immunostained with the following antibodies: goat anti-glutamate decarboxylase 67 (GAD 67; 1:500, 1:1000, 1:2000, 1:4000 dilutions; Chemicon), mouse (Ms) anti- glutamate receptor 2 (GluR2; 1:500 and 1:1000; NeuroMab), and Ms anti- metabotropic

glutamate receptor 1/5 (mGluR1/5; 1:500 and 1:1000; NeuroMab).

Epifluorescence microscopy. Images were acquired with a Leica DMI6000B epifluorescence microscope attached to a Hamamatsu Orca ER digital camera.

Results and DiscussionRetroviral vectors are injected intracranially to label precursor cells in the dentate gyrus.

Before I could begin contributing to our research project and forming my own research, I needed to learn how to perform all the necessary procedures with accuracy and timeliness. Because DGCs born after SE are more likely to have abnormalities, and thus morel likely to be the cause of seizures, we want to specifically identify cells based on their birthdate.

In order to birthdate DGCs we use a retroviral vector (RV) with a fluorescent reporter gene. For these experiments, we are using a synaptophysin-yellow fluorescent protein (syp-YFP) reporter. This is a fusion protein in which synaptophysin, a presynaptic terminal protein that is trafficked to the axon terminals, is conjugated to YFP. With this RV, the axons of DGCs are labeled with YFP and can be identified. When used in combination with antibodies that are specific to certain cell types, this approach allows us to see what types of cells DGCs are synapsing onto. However, these data are not shown in this proposal because, due to the experimental timeline, the animals I have injected with the syp-YFP are not ready for tissue processing. Instead, I am showing my pilot data indicating my successful RV administration.

RV is administered via intracranial injections into the dentate gyrus. This is a vital step in the experiment, because no data can be collected without accurate injections. Using a stereotaxic frame and pre-determined coordinates, I became adept at this procedure. I have successfully injected RV into the dentate gyrus of adult rats, both control and epileptic, hitting the center of the hilus between, the superior and inferior granule cell layers (Fig 1 A,B).

A

Hilus

GCL

Needle Track

B

HilusGCL

Figure 1. Two coronal sections of the same animal, showing one of my first retroviral (RV) hippocampal injections, displaying proficiency in location of injection site . A, Needle track (long arrow) can be seen going through granule cell layer (GCL) into hilus (H), where RV-GFP labeled cells (short arrow) are concentrated. B, A more rostral section from same animal. As expected, RV-GFP labeled cells (arrows) are DGC progenitors, limited to subgranular zone of GCL.

An antibody against mGluR1/5 did not successfully label interneurons in the dentate gyrus.In order to identify cells that may be post-synaptic to DGCs in both control and epileptic tissue, we are using a variety of antibodies that are known to label cell subtypes in the dentate gyrus (DG). A subtype of interneuron in the DG can be identified by their expression of metabotropic glutamate receptor 1 (mGluR1) (Acsády et al 1998). I piloted the use of this antibody in the lab with a concentration series, using a 1:500 dilution, a 1:1000 dilution, and a negative control. However, we did not see any positive staining in with either dilution (1:500 shown in Figure 2). It is important to note that the quality of images I was able to obtain with the Leica epifluorescence microscope does not allow for an appreciation of the detail present in the raw data. The tissue clearly shows a lack of signal. Currently I am not pursuing the use of this antibody because we have determined that the anti-GAD67 antibody is a

more promising way of identifying interneurons in the DG.

Antibodies against GAD67 label interneurons of the dentate gyrus.Glutamate decarboxylase 67 (GAD67) is the enzyme that catalyzes the reaction of glutamate to the inhibitory neurotransmitter GABA (Nouel et al 2012). Using an antibody directed against this protein, in conjunction with the syp-YFP RV, will allow us to identify inhibitory interneurons in the dentate gyrus and determine whether newborn DGCs are synapsing onto them. Normally, DGC axons synapse onto interneurons in the hilus. However, in TLE there is extensive interneuron cell death, and this loss of inhibitory interneurons may promote hyperexcitability in epilepsy.

Therefore, GAD67 can prove to be useful in tracking interneurons and their synaptic relationship with DGCs.

Four different dilutions of GAD67 were used and 1:500 was identified as the most promising (Figure 3).

Antibody against GluR2 labels hilar mossy cells in the dentate gyrus. Hilar mossy cells are glutamatergic interneurons in the dentate gyrus and can be identified with an antibody directed against glutamate receptor 2 (GluR2) (Henze et al 2007). The GluR2 receptor is only found in hilar mossy cells and not other interneurons, thus making the

antibody against the protein very specific. Under normal conditions, mossy cells receive input from DGCs, but it is not known whether this relationship is altered in TLE (Henze et al 2007). Therefore, using GluR2 in our rodent TLE model may offer insight into the reorganization of the dentate gyrus in epilepsy.

Figure 4. 20x magnification DG coronal section showing GluR2 positive mossy cells (arrows). Blue is bisbenzamide nuclear stain, green is GluR2

H

GCL

Figure 2. 20x Coronal section of dentate gyrus treated with mGluR1/5 antibody. No positive labeling was detected.

GCLHilus

Figure 3. Three interneuron cell bodies (arrows) labeled with GAD67 in the dentate gyrus under 40x magnification. Blue is bisbenzamide nuclear stain, and green is GAD67.

GCL

IML

Hilus

Two dilutions were tested and again the 1:500 dilution yielded stronger signals, though both dilutions showed labelef cells (Figure 4).

Conclusion and Further Research

In our ongoing attempt to understand the abnormalities of the dentate gyrus in TLE, we have developed a new RV reporter to specifically identify axon terminals of DGCs and elucidate their post-synaptic partners. In order to to better understand the axon structures and the connections they make, we have employed a host of antibodies to identify different cell types in the hilus. I tested out three different antibodies to see if they could identify the correct cell types with strong enough signals. mGluR1/5 antibody should label a receptor protein that is specific to interneurons of the dentate gyrus. However, the results showed no labeled cells so I will not pursue it further at the moment.

Conversely, anti-GAD67 showed a few labeled cells, but will require more work before I can use it for data quantification. With some refinement of the GAD67 antibody labeling procedures, I expect to be able to use this antibody to label interneurons in the rat DG. I will also continue to refine the GluR2 labeling protocol in order amplify the signal and reduce noise. Once the labeling protocols are refined for these antibodies, I will use the tissues collected from animals that I injected with the RV syp-YFP for double-label immunostaining. I will identify axon terminals with an antibody against YFP and their potential post-synaptic partners with the antibodies against GluR2 for mossy cells and GAD67 for inhibitory interneurons. We will then use a confocal microscope to confirm colabeling at a putative synapse for quantification.

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