Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Summary

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Abstract

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments ¹. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction ^2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation ^4-6.

Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.

These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits.

The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

Protocol

1. Lateral Fluid Percussion Injury

1. Anaesthetize the mouse using a mixture of ketamine and xylazine given intraperitoneally. Then prepare the mouse's head for incision using an iodine scrub.
2. Perform a craniectomy over the right parietal area using 3 mm (outside diameter) trephine.
3. Secure Luer-loc needle hub (inside diameter 3 mm) over the craniectomy using cyanoarylate and dental acrylic.
4. 24 hr later, anaesthetize the mouse using isoflurane via inhalation.
5. Once normal breathing resumes, but before the mouse becomes sensitive to stimulation, deliver 20 msec pulse of saline into the skull via the fluid percussion injury....

Discussion

Each technique outlined above contributes to the greater understanding of the underlying mechanism causing the observed behavioral deficit. By combining the unique information gained from each method we are able to examine the biological mechanisms with more precision.

Measuring fEPSPs is useful for quantifying the net synaptic efficacy of large, spatially defined regions of neurons. It also can provide information about the potential of a group of cells to undergo synaptic plasticity, a ne.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Axopatch 200B amplifier	Molecular Devices	AXOPATCH 200B	Patch-clamp rig
Digidata 1322A digitizer	Molecular Devices		Patch-clamp rig
MP-225 micromanipulator	Sutter	MP-225	Patch-clamp rig
DMLFSA microscope	Leica		Patch-clamp rig
Multiclamp 700B amplifier	Molecular Devices	MULTICLAMP 700B	Multipurpose (field) rig
Digidata 1440 digitizer	Molecular Devices		Multipurpos (field) rig
MPC-200 micromanipulator	Sutter	MPC-200	Multipurpose (field) rig
BX51WI microscope	Olympus	BX51WI	Multipurpose (field) rig
Axoclamp 900A amplifier	Molecular Devices	AXOCLAMP 900A	VSD rig
Digidata 1322 digitizer	Molecular Devices		VSD rig
Redshirt CCD-SMQ camera	Redshirt	NCS01	VSD rig
VT 1200S Vibratome	Leica	14048142066
P-30 Electrode puller	Sutter	P-30/P
cOmplete protease inhibitor	Roche	11697498001