Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

Summary

We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allows us to monitor activity in individual units of tethered cockroaches. Here we present a modified version of that technique that allows us to also record brain activity in freely moving insects.

Abstract

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform natural behavior. We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allowed us to record activity from multiple neurons simultaneously while a tethered cockroach turned or altered walking speed. While a major advance, tethered preparations provide access to limited behaviors and often lack feedback processes that occur in freely moving animals. We now present a modified version of that technique that allows us to record from the central complex of freely moving cockroaches as they walk in an arena and deal with barriers by turning, climbing or tunneling. Coupled with high speed video and cluster cutting, we can now relate brain activity to various parameters of the movement of freely behaving insects.

Introduction

This article describes a successful system for recording from neurons within the central complex (CC) of the cockroach, Blaberus discoidalis,  as the insect walks in an arena and deals with objects that cause it to turn around, tunnel under, or climb over obstacles. The wires can also be connected to a stimulator to evoke activity in the surrounding neuropil with consequent behavioral changes.

Over the last decade considerable attention has been directed at the roles played by various brain regions in controlling insect behavior. Much of this focus has been directed toward the midline brain neuropi....

Protocol

1. Preparation of Tetrode Wires

1. Pull out a very thin nichrome wire (12 μm diameter, PAC coating) of about 1.1 m length. Attach a tape tag to each end. Hang the wire over a horizontal threaded rod such that the two ends are at the same height near the benchtop.
2. Repeat step 1.1 for a second wire, making two more ends for a total of 4, and place it next to the first wire (about 1 cm in between).
3. Stick the four ends together with a tape tag and attach the tag to a motorized rotating winding device. This device can be made from an inexpensive dc motor.
4. Wind the tetrode in one direction for 2 min (60 rpm) and unwind it in the o....

Results

We recorded the neural activity of 50 units from the CC in 27 preparations for walking experiments. For 15 of those preparations (23 units), climbing experiments were also performed. Individual units are named according to preparation and unit numbers (e.g. ʻunit 1-2ʼ indicates preparation 1, unit 2).

Snapshots of the video of one climbing trial are shown in Figure 4. The entire video is available in supplemental Video 1 (The sound is from unit 1-2). .......

Discussion

While previous electrophysiological studies on the CC or other regions of the insect brain have provided us with insights into the central control of behavior, most of them were performed in either restrained preparations^9,11 or tethered ones^10,14. As a result, the animal’s sensory experience and physiological state could be very different from those in a natural setting. Furthermore, the behavioral tasks that the animal can perform are limited to one plane under those situations. Here we pres.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nichrome wire	Sandvik Heating Technology	Kanthal RO-800	Use for tetrode
Biomedical polyethylene tubing	A-M Systems	800700	Use for tetrode tubing
Lynx-8	Neuralynx		Use for multiunit recording
Cheetah 32	Neuralynx		Use for multiunit recording
High speed camera	Basler	A602f	Use for video recording for walking experiments
High speed camera	Casio	EX-FC150	Use for video recording for climbing experiments
WINanalyze	Winanalyze	version 1.4 3D	Use for video tracking
MATLAB	MathWorks	MATLAB R2012b	Use for TTL pulse generation and offline data analysis