Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

Summary

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

Abstract

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 µm in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

Introduction

Understanding the neural conduction or propagation of neural signals is crucial for determination of the mechanism of neural communication in both the normal function and pathological conditions in the brain ^1-3. The hippocampus is one of the most extensively studied structures in the brain since it plays fundamental role in several brain functions such as memory, and spatial tracking and is involved in several pathological changes that dramatically impact behavior as well ^1,6 . Although, the hippocampus exhibits a complex organization, the different elements of its structure can be readily identified and accessed in the slice preparation^4-6....

Protocol

NOTE: Animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the university. CD1 mice of either sex at the age of P10 to P20 are used in this study.

1. Solutions for Surgery and Experimental Recording

1. Prepare normal artificial cerebrospinal fluid (aCSF) buffer containing (mM): NaCl 124, KCl 3.75, KH₂PO4 1.25, MgSO₄ 2, NaHCO₃ 26, Dextrose 10, and CaCl₂ 2. Use this normal aCSF for tissue recovery after dissection, as well as for washing system at the beginning of the experiment.
2. Prepare sucrose aCSF that is used during the....

Results

The data shown in the figures here were recorded in the unfolded hippocampus preparation with 4-AP (100 µM) aCSF added during incubation of the tissue in the recording chamber at RT (25 °C). Normally activity starts within 5 min, but in some hippocampal tissues from the older animals it may take longer. The 4-AP-induced neuronal firing observed with the PMEA is the same as previously reported ^14,15. Since the electrodes have a height of 200 µm, the electrode tips are located just below the cell layer (.......

Discussion

The development of the unfolded hippocampus preparation, where the longitudinal and transverse axes of the hippocampus are preserved in combination with a penetrating microelectrode array, provides a powerful tool to investigate the anatomy connections or neural propagation in the hippocampus ⁷. This unfolding procedure is also applicable for studying hippocampus in adult mice. Recent studies with this preparation showed that the 4-AP-induced epileptiform activity could propagate with a diagonal wave front acr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
desiccator jar	LABRECYCLERS Inc.	5410	Place regular paper towels at the bottome of the jar for animal anesthesia use.
A blade and Custome made surgical stage for unfolding hippocampus	N/A	N/A	A petri dish is place upside down (in the center) in the ice with a wet filter paper place on top of it.
Custom made tissue recovery chamber	N/A	N/A	Plastic tubes were glued with plastic mesh at the bottom and bubbled with 95% O2/ 5% CO2 in the aCSF.
Straight Operating Scissors	Fisher Scientific	S17336B                                            Medco Instruments No.:81995	This scissors is used to   decapitate the mice.
Integra Miltex Goldman-Fox Scissors	Fisher Scientific	12-460-517                        MILTEX INC                           No.:5-SC-320	This scissors is used to cut the skull of the mice.
Miltex Hysterectomy Forceps	Claflin Medical equipment	CESS-722033-00001	This Forceps is used to peel the cut skull to expose the brain
Micro Spatula	Cardinal Health		This micro spatula is used to tranfer the whole brain of a semisphere into the recorering chamber.
Frey Scientific Stainless Steel Semi-Micro Spatula	Cardinal Health		this semi micro spatula is used to tranfer the unfolded hippocampus into the glucose aCSF in the recovering chamber.
small paint brush	Lowe's	tem #: 105657                  Model #: 90219	The one with the smallest size in a normal paint brush package
Fire polished glass help tool	N/A	N/A	This tool was fire polished and made from the regular Pasteur glass pipettes.
Custom made glass needle	N/A	N/A	This tool was fire polished and made from the regular Pasteur glass pipettes.
Custom made glass tool with a metal wire loop	N/A	N/A	This tool was fire polished and made from the regular Pasteur glass pipettes with a reshaped metal wire loop.
Custom made glass solution dropper	N/A	N/A	This tool was  made from the regular Pasteur glass pipettes with its tips cut and a rubber head attached with the cut end.
Custom made tissue anchor	N/A	N/A	Nylon fiber mesh was glued on a insulated copper wire ring. The tissue anchor was hold by an micromanipulator.
Custom fabricated microelectrode array	N/A	N/A	More detail about the array please refer to  Kibler, et al, 2011.
Custom made filter and amplifiers circuits for the array	N/A	N/A	More detail about the array please refer to  Kibler, et al, 2011.
Data acquisition processor 3400a	Microstar Laboratories	N/A	This is a complete data acquisition system with A/D converter.