The authors thank Nick Kathman for suggestions and help at preparing for the manuscript. This technique was developed in conjunction with work supported by the AFOSR under grant FA9550-10-1-0054 and the National Science Foundation under Grant No. IOS-1120305 to RER.

1. Preparation of Tetrode Wires
<ol>
	<li>Pull out a very thin nichrome wire (12 &#956;m 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.</li>
	<li>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).</li>
	<li>Stick the four ends together with a tape tag and attach the tag to a motorized rotating windi...

<ol>
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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 preparations9,11 or tethered ones10,14. As a result, the animal&#8217;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...

This article describes a successful system for recording from neurons within the central complex (CC) of the cockroach, Blaberus discoidalis, &#160;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 neuropils that are collectively referred to as the central complex (CC). Progress has been made as a result of wide varieties of techniques targeting questions about the role of the CC in behavior. Those techniques range from neurogenetic manipulations, primarily in Drosophila, coupled with behavioral analysis1-3, to electrophysiological techniques that monitor neural activity within the CC and attempt to relate that activity to behaviorally relevant parameters.&#160; &#160;

Electrophysiological&#160; techniques include intracellular recording from individual identified neurons4-9 and extracellular recording, often with multi channel probes10,11. These two techniques are complimentary. Intracellular recording with sharp electrodes or whole cell patch provides very detailed data on identified neurons, but is limited to one or two cells at once, requires limited or no movement, and can be maintained for relatively short periods of time. Extracellular recordings can be easily set up, do not require restraint, and can be maintained for hours. With multi channel tetrodes and cluster cutting, fairly large populations of neurons can be analyzed simultaneously9,12. While whole cell patch has been successfully used in tethered insects13, we feel that there is also a need for techniques that allow us to record neural activity in the brain for long periods of time in freely behaving insects as they deal with barriers to forward movement.

The need to record as the insect moves and bounces up and down pushed us toward extracellular recording methods. We have had good success recording in restrained preparations with commercially available 16 channels silicon probes11, however the small size of even large cockroaches means that the probes have to be mounted off the body. That, coupled with the delicacy of the probe tines, made them inappropriate for a free walking preparation. In two previous projects, we used bundles of fine wires forming a tetrode to accomplish similar recording properties but in a more robust arrangement. These tetrode bundles allowed us to record from tethered cockroaches and relate CC unit activity to changes in walking speed14 and turning behavior resulting from antennal contact with a rod10.

As useful as these tethered preparations have been and will continue to be, they do present some limitations. First, the behaviors that the insect can perform are limited to one plane. That is, we could readily evoke changes in walking speed or turning, but climbing and tunneling actions were not possible, at least with the typical tether arrangement. Second, our tethered preparations are &#8220;open loop&#8221;. That is, they do not allow for normal movement related feedback to the system. Thus, as the cockroach turned on our tether, its visual world was not altered accordingly. It is possible to build closed loop tether systems to introduce this kind of feedback. However, they are limited by the complexity of the programming and hardware of the simulated visual environment. Nevertheless, we felt that we could improve upon our existing tethered recording methods by recording from the animal as it walked freely in an arena or track and encountered objects as it would in its natural surroundings.

Although wireless systems for recording brain activity15 would be ideal, current systems have limitations in the number of recording channels, time of data acquisition, battery life and weight. We, therefore, opted to try to adapt our tethered recording system for use in freely moving preparations. As better wireless systems become available, this technique can be readily adapted to such devices. The system that is described in this article is light weight, works very well and appears to have little deleterious effect on the cockroach&#8217;s behavior. With an inexpensive high speed camera and cluster cutting software, activity in individual brain neurons can be related to movement. Here we describe the preparation of the tetrode wires and their implantation into the insect&#8217;s brain as well as recording techniques for electrical activity and motion and how those data can be brought together for subsequent analysis.

<table border="0" cellpadding="0" cellspacing="0" width="728">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Nichrome wire&#160;</td>
			<td style="width:179px;">Sandvik Heating Technology</td>
			<td style="width:137px;">Kanthal RO-800</td>
			<td style="width:205px;">Use for tetrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomedical polyethylene tubing</td>
			<td>A-M Systems</td>
			<td>800700</td>
			<td>Use for tetrode tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lynx-8</td>
			<td>Neuralynx</td>
			<td></td>
			<td>Use for multi-unit recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cheetah 32</td>
			<td>Neuralynx</td>
			<td></td>
			<td>Use for multi-unit recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High speed camera</td>
			<td>Basler</td>
			<td>A602f</td>
			<td>Use fir video recording for walking experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High speed camera</td>
			<td>Casio</td>
			<td>EX-FC150&#160;</td>
			<td>Use for video recording for climbing experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">WINanalyze</td>
			<td>Winanalyze</td>
			<td>version 1.4 3D</td>
			<td>Use for video tracking&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab</td>
			<td>MathWorks</td>
			<td>MATLAB R2012b</td>
			<td>Use for TTL pulse generation and off-line data analysis</td>
		</tr>
	</tbody>
</table>

extracellular wire tetrode recording in brain of freely walking insects

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.

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. &#699;unit 1-2&#700; 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 u...

Watch this Scientific Journal Video about Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects at JoVE.com

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

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.

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform ...

extracellular-wire-tetrode-recording-brain-freely-walking

Research

JoVE Journal

Neuroscience

16.4K visualizações.  Case Western Reserve University.  Nós já desenvolveu uma técnica para implantar fios tetrode no complexo central do cérebro de barata que nos permite monitorar a atividade em unidades individuais de baratas amarrados. Aqui apresentamos uma versão modificada do que a técnica que nos permite também gravar a atividade cerebral em insetos se movendo livremente. 