Role of Calcium in Seizure Activity

Sophia Mian & Renuka Ramanathan

Overview of Action Potential and Calcium's Role


Neurons communicate at the chemical synapse. As an action potential, or nerve impulse, travels down the presynaptic axon, it activates voltage gated Calcium channels. The influx of Calcium causes synaptic vesicles to release neurotransmitters into the synaptic cleft. These neurotransmitters bind to receptors on the postsynaptic neuron allowing the action potential to continue along the next axon. The diagram to the right shows the steps of propagating an action potential from one neuron to another. Image is taken from Immune dysregulation and self-reactivity in schizophrenia: Do some cases of schizophrenia have an autoimmune basis? Jones et. al. Immunology and Cell Biology (2005)

Past Research
Previous research has included the use of novel seizure models such as the Xenopus laevis, or tadpole to characterize the effect of developmental seizures on critical processes which underly neural circuit formation and assess long term functional consequences. The literature shows studies of the degradation of neuronal circuitry in a mouse brain with seizure activity. Pre-clinical trials have oftentimes revolved around obtaining a basis of inhibiting calcium influx (i.e. adding a calcium chelator) into the neuron as a method of treating seizures. Bearing in mind the current research being done in the field of seizure therapeutics, we would like to propose a method of combining technologies in order to obtain electrical activity and calcium level data, in order to directly compare the role of each in seizures.

Electrophysiology Recordings (EPR)
EPR enables us to study the electrical properties of cells by meausuring the voltage change due to electrical current flow. With respect to neurons, it allows researchers to directly measure the action potential activity. By attaching glass microelectrodes, researchers can obtain a readout of the voltage across a single neuron.

Two-Photon Calcium Microscopy
Two-Photon Calcium Imaging allows us to exploit calcium sensing methods in order to measure calcium levels in a cell. With respect to neurons, researchers will be able to analyze levels of calcium in a single cell and develop a connection between calcium and the action potential. By injecting a calcium sensitive dye and exciting this dye with a laser, researchers can analyze single cell responses to determine calcium levels.

Combined Technology Experimental Setup
Simultaneous electric potential and calcium spike measurements of single neurons will be obtained in a setup that combines EPR and TPM. The approach involves penetrating a neuron with a sharp glass electrode and using iontophoresis to inject the calcium dye - Fura-2. Fura-2 will be loaded into the neurons and the electrodes will create a current, causing like charges to repel. Thus, the direct current will drive, or pulse, Fura-2 into the neuron.

Research Proposal
The aforementioned techniques of EPR and TPM used independently give researchers great insight into the electrical activity and calcium levels within a neuron. While the calcium imaging and EPR give correlated data about seizure activity, no direct comparison between data sets can be drawn if these tests are conducted separately because as we said seizures cannot be replicated. The potential to obtain a more cohesive picture about what is happening during seizures and possibly create and evaluate therapies targeted at different points during the progression of neuron firing led us to search for a method to measure these valuable pieces of data in concert.

We hope to compare calcium levels and electrical activity collected during different seizure events in mice, to data collected simultaneously during one seizure event in one mouse, by using an experimental setup that fuses EPR and TPM.

Chemoconvulsant
Prior to beginning the main portion of our experiment, we will first need to determine the appropriate concentration (or more appropriately dose) of chemoconvulsant to use in a mouse model. Pentylenetetrazol (PTZ) is a commonly used chemoconvulsant in the literature. Hewapathirane et. al used 15 mM PTZ in order to induce a seizure in a Xenopus laevis tadpole. The first step in our protocol is to test a range of PTZ doses that will allow us to isolate a concentration that will give us behavior consistent with a seizure (this is characterized in the literature by excessive blinking of the eyes, myoclonic jerks, etc). Once we have agreed upon a concentration we can move to the actual experiment itself.

Defining the Sample
In order to test the effects (if any) of two-photon calcium imaging on EPR and vice versa, we will need two populations of mice that serve as controls and one population of mice that will serve as the test samples for our experiment. Group 1 mice will only follow the methods outlined for EPR. Group 2 mice will only follow the methods outlined for two photon microscopy. Group 3 mice will be apart of an experimental setup that combines both EPR and calcium imaging simultaneously. Ideally, in order to obtain results which are reliable and comparable, these populations of mice can be anywhere from 10-50.

Protocol for Anesthetizing & Inducing Seizures

 * 1) Anesthetize all three groups of mice. Possible anesthetics are ketamine/xylazine or urethane or alternatively, adult mice can be anesthetized by inhalation of isoflurane.
 * 2) Perform EPR on Group 1, Two-Photon Calcium Imaging on Group 2, & the combined technologies approach on Group 3 in order to record baseline neuronal activity.
 * 3) Paralyze mice (possibly with reversible paralytic agent pancuronium dibromide by injection)
 * 4) In Group 1
 * 5) Groups 2 and 3 – also load (directly into neurons) Fura-1, a calcium sensitive fluorescent indicator, in order to conduct two-photon calcium imaging
 * 6) Inject all three groups with PTZ in order to induce seizure
 * 7) *Inject Fura-2, a calcium sensitive dye, into the neurons of Groups 2 & 3
 * 8) Perform the three corresponding tests in order to record the seizure induced neuronal activity
 * 9) Inject an anti-epileptic drug (possibly valproate) in order to stop seizures

Part 3: Assessing the Reliability of Combined Technologies Setup
We must now assess if by combining the EPR and TPM images, we have somehow induced or magnified the seizure. In order to do this we will conduct TUNEL live/dead cell assay on all three groups of mice. If we have not in any way affected the seizure with our methods and if we have regulated the dose of PTZ injected into all the mice, we expect to see on average the same amount of cell death in our controls as in our experimental group.

TUNEL Live/Dead Cell Assay

 * 1) Determine size and area of slice to investigate
 * 2) Fixation of tissue
 * 3) *Fixation stabilizes microscopic cellular structures and compositions in the specimens to allow them to withstand subsequent processing and to preserve them for retrospective analyses. The fixed cell and tissue specimens can also be used to extract biosynthetic molecules for biochemical or nucleotide sequence analysis. Without fixation, it would be difficult, if not impossible, to sensitively detect, localize, and quantitate biosynthetic or environmental molecules in many kinds of cell and tissue specimens.
 * 4) Wash cells
 * 5) Label cells with Br-dUTP
 * 6) Rinse cells
 * 7) Stain cells with Stain cells with fluorescein-PRB-1 (a fluorescein labeled anti BrdU monoclonal antibody)
 * 8) Analyze via flow cytometry (also analyze a positive and negative control)

Positive Outcomes
If our TUNEL Assay shows statistically insignificant difference between our control and experimental data, then future researchers can use our methods to directly compare the action potential activity to level so of calcium in a neuron during a seizure event.

Negative Outcomes
If our TUNEL Assay shows statistically significant difference between our control and experimental data, then we will have to search for other methods to enable direct comparison of action potential to calcium levels. This may involve developing a mathematical model which will take into account the randomness of two different seizure events and produce comparable results.

Future Implications
Having a method by which to simultaneously measure two sets of data during a seizure event has great ramifications for the field of seizure research. Currently, seizure therapeutics research has considered the idea of finding a mechanism by which to inhibit the voltage gated calcium channels during the even of a seizure. In order to substantiate that methods of inhibition are working, one might imagine the application of our experimental setup. With the use of EPR + TPM, researchers will be able to validate that while action potential activity is being recorded, calcium levels are low within the same neuron. Thus, calcium inhibition must be occurring.

The unpredictability and randomness of seizures has caused it to be a very difficult topic to study and quantitatively assess. Our experiment allows for researchers to gain more stability and insight into the correlation between two separate events that occur in a seizure model.

Resources

 * Calcium imaging setup - laser and microscope
 * Electrophysiological recording setup
 * TUNEL assay kit
 * Flow cytometer
 * Large population of mice
 * Basic lab space
 * Basic lab chemicals