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Regional Differences in the Hippocampal Dentate Gyrus and CA1 stratum lacunosum-moleculare Regions Cause Different Amyloid Beta Production in C57BL/6J Mice After Kainic Acid Induced Seizures

Brett Palmero ’20, Andjela Dragojevic ’18, Emily Fink ’19, Philip Freund ’19, Nichole Hedger ’19
Department of Biology
Lake Forest College
Lake Forest, IL 60045

Abstract

 

Temporal lobe epilepsy (TLE) is a hippocampus- focused subtype of epilepsy. The hallmark of all epileptic diseases is the seizure, which is caused by excessive synaptic activity in the brain. High synaptic activity has been correlated with an increase in Amyloid-β (Aβ) production, a hallmark of Alzheimer’s disease. The TLE-Aβ connection was examined in this study using a kainic acid (KA)-induced rodent model of epilepsy and immunohistochemical staining. The regions studied were the dentate gyrus (DG) mo and CA1 slm of the hippocampus. No significant difference in Aβ expression was found between KA and SA (control) conditions for both the Ca1 slm and DG mo regions. However, a significant increase in optical density in the KA CA1 slm region was found when compared to the KA DG mo region. This research will help target areas of the hippocampus for the treatment of Alzheimer’s disease in TLE patients.

 

Introduction

 

One of the most debilitating neurological diseases in the United States is epilepsy (Milligan, 2017). Epilepsy is characterized by one or more seizures in the afflicted person. A seizure begins with irregular electrical activity within the brain which can cause motor impairment and disorientation for a few seconds to a few minutes. These seizures can lead to distress and impairment for the afflicted person and can lead to a decrease in quality of life, especially if a patient experiences multiple seizures a day. The epilepsy of interest in this study was the temporal lobe epilepsy (TLE) subtype. TLE is focused within the temporal lobe, specifically the hippocampus. In humans, a seizure caused by this type of epilepsy is characterized by abnormal sensations such as déjà vu or nausea. These sensations are followed by disorientation and confusion. Seizures can be induced using kainic acid (KA) for research purposes (Stafstrom, Thompson, and Holmes, 1992). One possible connection to TLE is an increased risk of Alzheimer’s disease. In Alzheimer’s, the brain is plagued with Amyloid-β (Aβ) plaques. These plaques consist of Aβ proteins that come out of solution and coagulate, causing damage to surrounding neurons. Aβ is created when Amyloid precursor protein (APP) is snipped. The snipped fragment is usually degraded within the brain, but if a corrupt form of APP is snipped, Aβ can be the resulting fragment. (Klementieva et al., 2017). Aβ has been found in high concentration in the hippocampus of TLE patients (Sheng, Boop, Mrak, and Griffin, 1994). APP mRNA levels have been shown to be increased in the hippocampus when a seizure was induced using KA (Willoughby et al., 1992). Additionally, research has identified a positive correlation between Aβ expression and synaptic activity (Cirrito, 2005). An inhibition of synaptic activity in the dentate gyrus (DG) region of the hippocampus results in a decrease in Aβ . The DG is the focus of this study because it’s high synaptic density is believed to lead to increased Aβ levels and, ultimately, neuronal death. Hippocampal areas such as the CA1 stratum lacunosum-moleculare (CA1 slm) are interneuron heavy and have fewer synapses, so these areas are expected to have lower Aβ production than areas in the DG (Kunkel et al., 1988). The hypothesis of this study was that induced seizures cause increased Aβ production in the DG due to the high number of synaptic connections. The combination of increased seizure-induced synaptic activity and the high number of synapses in these regions may lead to a significant increase in Aβ deposit. This would reveal areas for treatment to prevent Alzheimer’s disease in TLE patients.

 

Materials and Methods

 

Brain Slice Preparation:

C57BL/6J mice were injected with KA or saline (SA) as a control in one hemisphere at P22 to P30 days after birth. The mice were sacrificed 7 days later. The mice were perfused, and their brains extracted, cut into 100 µM sections, and stored in cryoprotectant.

 

IHC Day 1:

The IHC procedure was provided by Professor Hedrick. (Hedrick, 2017). Slices were washed with phosphate buffered saline (PBS) to remove the cryoprotectant. They were then incubated in blocking serum. The blocking solution consisted of 0.5% triton, 5% bovine serum albumin (BSA), and 5% goat serum in PBS. Once incubated, they were put into overnight washes of the appropriate 3D6 primary antibody concentration.

 

IHC Day 2:

Slices were washed with blocking serum and then put into a 1:2,000 goat anti-mouse dilution of secondary antibody incubation. The slices were washed in PBS and 0.3% H2O2. Lastly, the slices were washed in PBS again overnight.

 

IHC Day 3:

The slices were incubated in avidin-biotin complex (ABC) kit. The ABC was made up 30 minutes before use, using 100 µL avidin, 100 µL biotin, and 10 mL of PBS. This was followed by a 0.05% molar tris-buffered saline (TBS) solution for 10 minutes. Then the slices were put into diaminobenzidine (DAB) solution until the sections took on a light colour. The DAB solution was made up 10 minutes before use, using 15 mL ddH2O, 1 gold DAB tablet, and 1 silver DAB tablet. TBS solution washes were repeated and then the slices were mounted with no coverslip to dry in a drawer overnight.

 

IHC Day 4:

The mounted slides were put into ethanol washes and deionized H2O (ddH2O) washes. They were taken out and put into Cresyl Violet for 10 minutes. Next, the ethanol and ddH2O washes were repeated. Finally, they were coverslipped with glycerol-based mounting medium Mowiol and left to sit for at least 24 hours to allow for drying until imaging.

 

Imaging:

Each slice was imaged and then stitched using ImageJ. The quantitative analysis was done using Fiji. Aβ expression was quantified by the calculation of regional optical density. The dentate gyrus granule cell layer (DG sg) region was used for background. It was subtracted from CA1 slm  and dentate gyrus molecular level (DG mo). The regions were compared using an ANOVA.

 

IHC Run 1:

The first IHC run had 1 KA mouse brain slice in a 1:1,000 and 1 in a 1:3,000 3D6 anti-Aβ primary antibody dilution, and 1 as a no primary antibody control. There was also 1 SA slice in a 1:1,000 and 1 in a 1:3,000 3D6 primary antibody dilution. The purpose of this run was to optimize the concentration of primary antibody to yield more specific staining, with less background.

The second run through was used to replicate results for analysis. It had 6 KA slices in 1:3000 3D6 primary antibody dilution and 3 SA slices in 1:3000 3D6 primary antibody dilution.

 

IHC Run 2:

After running the initial staining procedure using 5 slices of varying primary antibody concentrations, it was determined that the 1:3,000 3D6 primary antibody dilution gave the best stain (Figure 1). The remaining 9 of the slices were stained using the 1:3,000 3D6 primary antibody dilution. Due to the lack of no antibody control in the second run, the only no antibody control used was from the first run. The layers of interest after DAB staining are labeled in Figure 2. The stain comparisons of the cresyl violet and DAB are seen in Figure 3. DG sg was used as a background due to high cell body density. The DG mo and CA1 slm were measured and compared in Figure 4.

 

ANOVA:

ANOVA was used to analyze the KA and SA conditions and the CA1 slm and DG mo regions. ANOVA was used because it is useful for studying regional differences between the CA1 slm and the DG mo while comparing the KA and SA conditions. It also can reveal any interactions between the 2 independent variables.

 

Results:

 

The (Brain region: CA1 slm vs. DG mo) X (Condition: KA vs SA) ANOVA showed no significant difference between SA and KA conditions for both the CA1 slm and the DG mo. KA brain slices (M=.245) had similar optical densities to SA brain slices (M=.240), F(1, 23)=.038, p=.846. The graph is shown in Figure 4. This means there is not enough evidence to support our hypothesis.

 

The ANOVA found an interaction, F(1, 23)=4.346, p=.048, that showed a significant difference between the DG mo and CA1 slm regions in the KA condition. The CA1 slm (M=.281) had significantly higher optical densities than the DG mo (M=.204), F(1, 23)=7.962, p=.01. This is contradictory to the proposed hypothesis since the CA1 slm was believed to produce less Aβ post seizure. The post hoc tests found that in the KA treated brains, the CA1 slm region (M=.312) had significantly higher optical densities than the DG mo region (M=.179), but in the SA brains, there was no significant difference in optical densities between the CA1 slm region (M=.250) and the DG mo region (M=.230). This interaction shows that the CA1 slm and DG mo produced similar amounts of Aβ before the seizures. When the seizure was induced, the CA1 slm created significantly more Aβ than the DG m0 in response to the increased neuronal activity.

 

Discussion:

 

KA and SA Conditions:

The results revealed no significant difference between KA and SA optical density for both CA1 slm and DG mo. Thus, sufficient evidence to support a significant increase in Aβ after KA-induced seizures is lacking. Since this is the case, the proposed hypothesis was rejected. The DG mo layer was focused on because it is known to have abundant synapses which was hypothesized to have greater ability to produce Aβ in a seizure. Despite this, there was no significant difference between the KA and SA conditions. This could be due to the low number of slices measured. The graphs showed a visible difference between the two conditions, with trends going both ways, so further study needs to be done.

 

CA1 slm Region:

The CA1 slm region had the most visible Aβ staining. The analysis between the KA and SA conditions revealed no significant difference, but when both DG mo and slm regions were compared in the KA condition, the CA1 slm region had a significantly higher level of staining than the DG mo region. When the two regions were compared in the SA condition, there was no significant difference. The lack of significant regional difference in the SA condition compared to the significant regional difference in the KA condition highlights possible differences in neuronal organization in the CA1 slm that would account for the Aβ difference post seizure. This region is known to have interneurons and few synapses, in contrast to the high number of synapses in the DG mo (Kunkel et al., 1988). This counters the proposed hypothesis that heavy synaptic areas produce more Aβ. There is most likely a mechanism that occurs in the neuron cell body, axons, or dendrites that increases Aβ production depending on the amount of neuronal activity. The evidence from this study supports that neuronal activity influences Aβ production, but the focus of the study should shift from synaptic activity to overall neuronal activity. Studying areas that have larger numbers of cell bodies, axons, and dendrites such as the CA1 slm would provide better insight to Aβ production post seizure.

 

Possible Endocytic Mechanism in Aβ Release:

APP is known to be excised and either internalized or externalized. When APP is internalized via endocytosis, Aβ has been found to increase in the interstitial fluid (Cirrito, 2008). This endocytic pathway has been shown to be located closer to the cell body, so lysosomes are able to more easily degrade the incoming APP (Koo et al,  1996). This pathway is abundant in CA1 slm regions because of the copious interneuronal bodies, which could be why there was a significant increase in Aβ in these regions when compared to the KA DG mo. If this pathway were dependent on synaptic activity, the DG mo region would have had a higher amount of Aβ. Since a significantly higher amount of Aβ was present in CA1 slm regions post seizure, this pathway is most likely dependent on overall neuronal activity. The CA1 slm is more susceptible to increased Aβ and the Alzheimer’s pathology than the DG mo region, according to this study’s findings.

 

Methodology Improvements:

Replications should be completed in further studies. There was a trend in the data that supported the hypothesis, but there was no significance. The lack of significance could be due to the low number of brain slices used for analysis (14). With repetition, more power will be added to the study, increasing the ability to find any significant difference between the KA and SA conditions. Replications with other animal antibodies should be included because the secondary antibody used in this study was goat anti-mouse. Using a mouse antibody is not the best for specific staining when working on mouse brains because it could bind to antibodies other than Aβ. Another animal’s antibody would be more specific when finding the protein of interest. Lastly, the replications should be done on mice that have the induced seizures for a longer time. This means letting them live more than 7 days before sacrificing them while inducing more seizures. Any difference in Aβ production would then be more evident since the brains would have more time to accumulate the protein.

 

Region Sensitivity and Focused Treatment:

Another important improvement is to switch focus to regions like the CA1 slm. This region consistently had dark staining and significantly more Aβ than the DG mo region. The interaction between the two regions highlights regional differences that can cause different susceptibilities to seizure activity in terms of Aβ production. The regional difference found in this study showed that  Aβ may be released near heavy cell body, axonal, and dendritic regions instead of heavy synaptic regions. The mentioned endocytic pathway may explain why the CA1 slm region was more sensitive to seizure activity than the synapse heavy DG mo region. Studying different regional sensitivity will help prevent Alzheimer’s for someone who already has TLE.

 

Concluding Thoughts:

While no significant evidence was found to support the proposed hypothesis, trends emerged in the data that may lead to more significant findings in further studies. The CA1 slm also offered new insight into a possible mechanism that produces Aβ and is overactivated during a seizure. Overall this study has offered insight into what causes the connection between Alzheimer’s and TLE.

 

Acknowledgements:

 

Thank you to Dr. Tristan Hedrick for supplying us with KA and SA-injected mice brain sections, and for instructing and advising us throughout our experimental procedure and write-up. Thank you to Schuyler Kogan for also guiding us as well. Thanks to the Northwestern University Robert Vassar Lab for supplying us with the Novus Biologicals 3D6 Aβ primary antibody. Thanks to Yoan Ganev for data analysis advice.

 

References:

 

Cirrito, J. R., Kang, J. E., Lee, J., Stewart, F. R., Verges, D. K., Silverio, L. M., … & Holtzman, D. M. (2008). Endocytosis is required for synaptic activity-dependent release of amyloid-β in vivo. Neuron, 58(1), 42-51.

 

Cirrito, J. R., Yamada, K. A., Finn, M. B., Sloviter, R. S., Bales, K. R., May, P. C., … & Holtzman, D. M. (2005). Synaptic activity regulates interstitial fluid amyloid-β levels in vivo. Neuron, 48, 913-922.

 

Hedrick, T. P., Nobis, W. P., Foote, K. M., Ishii, T., Chetkovich, D. M., & Swanson, G. T. (2017). Excitatory Synaptic Input to Hilar Mossy Cells under Basal and Hyperexcitable Conditions. eNeuro, 4(6), ENEURO-0364.

 

Ikeda, S., Allsop, D., & Glenner, G. G. (1989). Morphology and distribution of plaque and related deposits in the brains of Alzheimer’s disease and control cases. An immunohistochemical study using amyloid beta-protein antibody. Laboratory investigation; a journal of technical methods and pathology, 60(1), 113-122.

 

Klementieva, O., Willén, K., Martinsson, I., Israelsson, B., Engdahl, A., Cladera, J., … & Gouras, G. K. (2017). Pre-plaque conformational changes in Alzheimer’s disease-linked Aβ and APP. Nature communications,14726.

 

Koo, E. H., & Squazzo, S. L. (1994). Evidence that production and release of amyloid beta-protein involves the endocytic pathway. Journal of Biological Chemistry, 269(26), 17386-17389.

 

Koo, E. H., Squazzo, S. L., Selkoe, D. J., & Koo, C. H. (1996). Trafficking of cell-surface amyloid beta-protein precursor. I. Secretion, endocytosis and recycling as detected by labeled monoclonal antibody. Journal of cell science, 109(5), 991-998.

 

Kunkel, D. D., Lacaille, J. C., & Schwartzkroin, P. A. (1988). Ultrastructure of stratum lacunosum moleculare interneurons of hippocampal CA1 region. Synapse, 2(4), 382-394.

 

Mackenzie, I. R., & Miller, L. A. (1994). Senile plaques in temporal lobe epilepsy. Acta neuropathologica, 87(5), 504-510.

 

Milligan, T. A. (2017). IS EPILEPSY THE DIAGNOSIS?.

  

Priller, C., Bauer, T., Mitteregger, G., Krebs, B., Kretzschmar, H. A., & Herms, J. (2006). Synapse formation and function is modulated by the amyloid precursor protein. Journal of Neuroscience, 26(27), 7212-7221.

 

Sheng, J. G., Boop, F. A., Mrak, R. E., & Griffin, W. S. T. (1994). Increased Neuronal β‐Amyloid Precursor Protein Expression in Human Temporal Lobe Epilepsy: Association with Interleukin‐1α Immunoreactivity. Journal of neurochemistry, 63,, 1872-1879.

 

Stafstrom, C. E., Thompson, J. L., & Holmes, G. L. (1992). Kainic

 

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Figure Text:

Figure 1. Initial Staining with no antibody control (SA (-)), 1:1,000 primary antibody dilution (SA 1:1,000), and 1:3,000 primary antibody dilution for the SA condition (SA: 1:3,000) and the KA condition (KA 1:3,000). The KA 1:3,000 dilution shows the most antibody, indicating more Aβ than the controls.  Since it also showed the best staining, it was used for the replications.

 

Figure 2. The hippocampal regions of interest are shown with DAB staining. The CA1 stratum lacunosum moleculare (slm) shows more staining when compared to the dentate gyrus molecular layer (DG mo). The dentate gyrus granule cell layer (DG sg) is to be used as background for the because of its very light staining. The DG sg will be subtracted from the DG mo and CA1 slm to calculate their relative optical densities.

Figure 3. The cresyl violet stain was used to stain for cell bodies. It allowed for the regions to be differentiated by cell body concentration. The DAB staining showed where the 3D6 antibody bound to Aβ. Both SA (SA-Cresyl violet and SA-DAB) and KA (KA-Cresyl violet and KA-DAB) conditions were highlighted. For an idea of what both of these stains look like layered on top of one another, reference Figure 1.

Figure 4. There was no significant difference between the KA and SA conditions, F(1, 23)=.038, p=.846. There however was an interaction, F(1, 23)=4.346, p=.048. The CA1 slm region had significantly higher Aβ than the DG mo in the KA condition, (M=.204), F(1, 23)=7.962, p=.01. There was no significant difference between the CA1 slm and DG mo in the SA condition.

 

Primary Antibody

Figure 1. Initial Staining with no antibody control (SA (-)), 1:1,000 primary antibody dilution (SA 1:1,000), and 1:3,000 primary antibody dilution for the SA condition (SA: 1:3,000) and the KA condition (KA 1:3,000). The KA 1:3,000 dilution shows the most antibody, indicating more Aβ than the controls. Since it also showed the beststaining, it was used for the replications.

Figure 1. Initial Staining with no antibody control (SA (-)), 1:1,000 primary antibody dilution (SA 1:1,000), and 1:3,000 primary antibody dilution for the SA condition (SA: 1:3,000) and the KA condition (KA 1:3,000). The KA 1:3,000 dilution shows the most antibody, indicating more Aβ than the controls. Since it also showed the beststaining, it was used for the replications.

 

Figure 1. Initial Staining with no antibody control (SA (-)), 1:1,000 primary antibody dilution (SA 1:1,000), and 1:3,000 primary antibody dilution for the SA condition (SA: 1:3,000) and the KA condition (KA 1:3,000). The KA 1:3,000 dilution shows the most antibody, indicating more Aβ than the controls.  Since it also showed the beststaining, it was used for the replications.

Hippocampal Regions of Interest

Palmero Figure 2

 

Palmero Figure 2

 

Figure 2. The hippocampal regions of interest are shown with DAB staining. The CA1 stratum lacunosum moleculare (slm) shows more staining when compared to the dentate gyrus molecular layer (DG mo). The dentate gyrus granule cell layer (DG sg) is to be used as background for the because of its very light staining. The DG sg will be subtracted from the DG mo and CA1 slm to calculate their relative optical densities.

 

Cresyl Violet and DAB Staining Comparisons

Palmero Figure 3

 

Figure 3. The cresyl violet stain was used to stain for cell bodies. It allowed for the regions to be differentiated by cell body concentration. The DAB staining showed where the 3D6 antibody bound to Aβ. Both SA (SA-Cresyl violet and SA-DAB) and KA (KA-Cresyl violet and KA-DAB) conditions were highlighted. For an idea of what both of these stains look like layered on top of one another, reference Figure 1.

 

Brain Region and Condition Mean Optical Density Comparisons

Palmero Figure 4

 

Figure 4. There was no significant difference between the KA and SA conditions, F(1, 23)=.038, p=.846. There however was an interaction, F(1, 23)=4.346, p=.048. The CA1 slm region had significantly higher Aβ than the DG mo in the KA condition, (M=.204), F(1, 23)=7.962, p=.01. There was no significant difference between the CA1 slm and DG mo in the SA condition.

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