Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Summary

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

Abstract

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham "no seizure" rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein α-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Introduction

Electroconvulsive therapy has been used to treat patients with major depressive disorders, including severe drug-resistant depression, bipolar depression, Parkinson's diseases, and schizophrenia^1,2. In this therapy, seizure is generated by electrical stimulus delivered to the head of anesthetized patients via epicranial electrodes^1,2,3. Repetitive administration of ECS has been clinically beneficial to drug-resistant depressive disorders^1,2,

Protocol

All experimental procedures including animal subjects have been approved by the Institutional Animals Care and Use Committee at the University of Illinois at Urbana-Champaign.

1. Maintaining a Rat Colony

1. Breed Sprague-Dawley rats (see the Table of Materials) and maintain them in standard conditions with a 12-h light-dark cycle and ad libitum access to food and water.
2. Wean the rat pups at postnatal day (P) 28 and house them in groups of 2 - 4 male or female littermates.
3. Mark the tails of male rats with a non-toxic permanent black marker for identification.
4. Weigh the male rats 3 times....

Results

Using the detailed procedure presented here, one electrical shock (55 mA, 100 pulses/s for 0.5 s) delivered via ear-clip electrodes induced nonrecurring stage 4-5 tonic-clonic seizures in rats (Figure 1A-B). A total 8 of rats received acute ECS induction and displayed stage 4-5 tonic-clonic seizures. The seizures lasted about 10 s, and all rats recovered within 1 - 2 min of seizure cessation. Sham "no seizure" rats did not receive an electric.......

Discussion

Here, we describe an ECS induction method in rats that elicits the global stimulation of neuronal activity in their hippocampi. ECS is an animal model of electroconvulsive therapy, which is clinically used to treat drug refractory depressive disorders in humans^1,2,3. Despite use of electroconvulsive therapy to treat severe depression, the precise underlying mechanism remains unclear. Because ECS induces anti-depressant-like beha.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Spargue-Dawley rat	Charles River Laboratories		ECS supplies
A pulse generator	Ugo Bsile, Comerio, Italy	57800	ECS supplies
MilliQ water purifying system	EMD Millipore	Z00Q0VWW	Subcellular fractionation supplies
Sucrose	Em science	SX 1075-3	Subcellular fractionation supplies
Na4O7P2	SIGMA-ALDRICH	221368	Subcellular fractionation supplies
Ethylenediaminetetraacetic acid (EDTA)	SIGMA-ALDRICH	E9884	Subcellular fractionation supplies
HEPES	SIGMA-ALDRICH	H0527	Subcellular fractionation supplies
Okadaic acid	TOCRIS	1136	Subcellular fractionation supplies
Halt Protease Inhibitor	Thermo Scientific	78429	Subcellular fractionation supplies
NaVO3	SIGMA-ALDRICH	72060	Subcellular fractionation supplies
EMD Millipore Sterito Sterile Vacuum Bottle-Top Filters	Fisher Scientific	SCGPS05RE	Subcellular fractionation supplies
Iris Scissors	WPI (World Precision Instruments)	500216-G	Subcellular fractionation supplies
30 mm tissue culture dish	Fisher Scientific	08-772B	Subcellular fractionation supplies
Glass homogenizer and a Teflon pestle	VWR	89026-384	Subcellular fractionation supplies
1.7 mL microcentrifuge tube	DENVILLE SCIENTIFIC INC.	C2170 (1001002)	Subcellular fractionation supplies
Sorvall Legend XT/XF Centrifuge	Thermo Fisher	75004521	Subcellular fractionation supplies
Pierce BCA Protein Assay Reagent A, 500 mL	Thermo Fisher	#23228	Western blot supplies
Pierce BCA Protein Assay Reagent B, 25 mL	Thermo Fisher	#1859078	Western blot supplies
SDS-polyacrylamide gel (SDS-PAGE)	BIO-RAD	#4561086S	Western blot supplies
Running Buffer	Made in the lab		Western blot supplies.
Mini-PROTEAN Tetra Vertical Electrophorsis Cell for MiniPrecast Gels, 4-gel	BIO-RAD	#1658004	Western blot supplies
Polyvinyl difluoride (PVDF) membrane	Milipore	IPVH00010	Western blot supplies
Transfer Buffer	Made in the lab		Western blot supplies.
Tris-base	Fisher Scientific	BP152-1	Western blot supplies
Glycine	Fisher Scientific	BP381-5	Western blot supplies
Sodium dodecyl sulfate	SIGMA-ALDRICH	436143	Western blot supplies
Methanol	Fisher Scientific	A454-4	Western blot supplies
Triton X-100	Fisher Scientific	BP151-500	detergent for PSD isolation
Mini Trans-Blot Module	BIO-RAD	#1703935	Western blot supplies
Nonfat instant dry milk	Great value		Western blot supplies
Multi-purposee rotator	Thermo Scientific	Model-2314	Western blot supplies
Hyblot CL Autoradiography Film	DENVILLE SCIENTIFIC INC.	E3018 (1001365)	Western blot supplies
Enhanced chemifluorescence substrate	Thermo Scientific	32106	Western blot supplies
a Konica SRX-101A film processor	KONICA MINOLTA	SRX-101A	Western blot supplies
Name of Antibody
PSD-95	Cell Signaling	#2507	Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Rabbit
Synaptophysin	Cell Signaling	#4329	Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Rabbit
alpha-Tubulin	Santacruz	SC-5286	Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Mouse
GluN2B	Neuromab	75-097	Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Mouse
GluA2	Sigma-aldrich	Sab 4501295	Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Rabbit
STEP	Santacruz	SC-23892	Antibody dilution = 1:200 - 500, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Mouse
Peroxidas AffiniPure Donkey Anti-Mouse IgG (H+L)	Jackson ImmunoReserch laboratory	715-035-150	Antibody dilution = 1:2,000-5,000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey
Peroxidas AffiniPure Donkey Anti-Rabbit IgG (H+L)	Jackson ImmunoReserch laboratory	711-035-152	Antibody dilution = 1:2,000-5,000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey