Role of Calcium in Seizure Activity

Sophia Mian & Renuka Ramanathan

Background

Overview of Action Potential and Calcium's Role

Propagation of Action Potential at the Synaptic Cleft Influx of Calcium allows for nerve impulses to jump from one neuron to the next.

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.

Research Proposal

•Currently, there is little information on the role of Calcium in a brain with seizure activity

•Studies have been done showing that a calcium spike is observe in neurons within a seizure model and also that these spikes are not seen in a brain exhibiting normal behavior

•Our goal is to further study the role of Calcium by investigating the influx of Calcium into the pre-synaptic terminal (i.e. is the voltage gated Calcium channel constitutively open?) over time.

•Our methods will include the use of a calcium indicator dye (the same as used in the Okhi article) and two photon microscopy to reveal the presence of Calcium

Methods

Part 1: Determining the Dose

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.

Part 2: Experimental Setup & Methods

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

References

Two-photon Imaging of Synaptic Plasticity and Pathology in the Living Mouse Brain

Grutzendler et. al.

Two-photon microscopy is used to study the neuronal structure of animal models of neurodegeneration, brain injury and cerebrovascular disease

Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex

Ohki et. al.

Employ calcium sensing to reveal the micro-architecture in the visual cortex of the brains of rats and cats

In vivo imaging of seizure activity in a novel developmental seizure model

Hewapathirane et. al.

Characterize an in vivo model of seizures in Xenopus laevis tadpole – allowing direct examination of seizure activist and seizure induced effects on neuronal development