Name: a procedure for implanting organized arrays of microwires for single-unit recordings in awake, behaving animals
Uploaded: 2014-02-14T14:15:00
Description: Watch this Scientific Journal Video about A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals at JoVE.com

This study was supported by the National Institute on Drug Abuse grants DA 006886 (MOW) and DA 032270 (DJB).

The utmost care must be taken to maintain aseptic conditions (as described in the Guide for the Care and Use of Laboratory Animals9) while preparing for and conducting the following procedure. The following protocol is in compliance with the Guide for the Care and Use of Laboratory Animals and has been approved by the Institutional Animal Care and Use Committee, Rutgers University. It is estimated that the subsequent procedures will require 3-6 hr to complete.
Implanting the Microwire ...

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Extracellular recordings represent a powerful experimental technique that can be incorporated into nearly any experimental preparation in neuroscience. Wires that have been implanted in organized arrays can be tracked as their shafts pass through the brain and into their target region (Figure 5A). When a small, post-experimental lesion is created at the noninsulated microwire tip to create a small iron deposit from the stainless steel wire, one can precisely mark the location of the uninsulated microwire...

Electrophysiological recordings allow scientists to examine the electrical properties of biological cells. In the central nervous system, where electrical impulses serve as a signaling mechanism, these recordings are of particular importance for understanding neural function1-2. During single-unit recordings in behaving animals, a microelectrode that has been inserted into the brain is able to record changes in a neuron&#39;s generation of action potentials over time.
While many techniques allow one to record brain activity, single-unit electrophysiology is one of the most precise methods by allowing resolution at the single neuron level. When a high degree of spatial specificity is desired, microwires can be used to target discrete sub-nuclei or ensembles of cells within the brain3. Single-unit recordings also benefit from high temporal resolution as recordings are accurate at the microsecond level. And, in vivo awake recordings allow intact circuit interactions, with the natural milieu of afferent and efferent projections, systemic chemical and hormonal influences, and physiological parameters. Neural signals are derived from sensory input, motor behaviors, cognitive processing, neurochemistry/pharmacology, or some combination. Accordingly, the segregation of sensory, motor, cognitive, and chemical influences necessitates well-conceived experiments with effective contingencies and controls that may allow for the assessment of each of the aforementioned influences. All in all, recordings in behaving animals allow experimenters to observe the integration of multiple sources of information within a functioning circuit and to derive a more comprehensive model of circuit function.
Single-unit recordings also suffer from a number of disadvantages of which any experimenter should be aware. First and foremost, recordings can be difficult to conduct. Indeed, properties of the headstage amplifiers and the implanted microwires that allow for spatial and temporal specificity in these recordings also makes recordings susceptible to the influence of extraneous electrical signals (i.e. electrical &#34;noise&#34;). Accordingly, the ability to troubleshoot problems in an electrophysiological system necessitates a well-developed technical understanding of electrophysiological principles and apparatus. It is also important to note that, under certain circumstances, recorded electrical signals in extracellular recordings can represent the summation of multiple neural signals. Moreover, the generalizability of single-unit activity to population activity within a target region can often be limited by the degree of cellular heterogeneity within the target region (but see Cardin4). For example, electrodes might be biased towards recording high amplitude output neurons in lieu of other cells. The interpretability of single-unit recordings is increased by combining recordings with other techniques including, but not limited to, electrical (orthodromic or antidromic), chemical (e.g. iontophoretic or designer receptor) or optogenetic stimulation4, temporary neural inactivations, sensorimotor examinations5, disconnection procedures, or immunohistochemistry3.
In the protocol that follows we will enumerate the materials and steps necessary to implant an organized microwire array in the rat (although the protocol can be adapted for use in other species). The procedure and style of fixed arrays used in our laboratory have proven reliable for longitudinal recordings and can sustain recordings of the same neuron for over one month&#39;s time 6-8. This makes this procedure ideal for examining phasic responses to experimental stimuli, plastic changes in neural responses, or mechanisms of learning and motivation.

<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Table 1. List of Surgical Materials</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Fisher (MooreBrand)</td>
			<td>19-898-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Swabs</td>
			<td>Fisher (Puritan)</td>
			<td>S304659</td>
			<td></td>
		</tr>
		<tr>
			<td>Nembutal (Pentobarbital)</td>
			<td>Sigma Aldrich</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Methyl Nitrate</td>
			<td>Sigma Aldrich</td>
			<td>A0382</td>
			<td></td>
		</tr>
		<tr>
			<td>Baytril (Enrofloxacin)</td>
			<td>Butler Shein (Bayer)</td>
			<td>1040007</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride</td>
			<td>Butler Shein</td>
			<td>SKU# 023061</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine (Povidone-Iodine)</td>
			<td>Fisher (Perdue)</td>
			<td>19-066452&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotax</td>
			<td>Kopf</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>Cauterizing Tool</td>
			<td>Stoelting</td>
			<td>59017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Nikon</td>
			<td>SMZ445</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental Drill</td>
			<td>Buffalo</td>
			<td>37800</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriostatic Saline</td>
			<td>Bulter Schein</td>
			<td>8973</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewlers Skrews</td>
			<td>Stoelting</td>
			<td>51457</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwire Array</td>
			<td>Microprobes</td>
			<td>Custom (Flexible)</td>
			<td></td>
		</tr>
		<tr>
			<td>Ground Wire</td>
			<td>Omnetics</td>
			<td>Custom Plug</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental Acrylic</td>
			<td>Fisher (BAS)</td>
			<td>50-854-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbable Sutures</td>
			<td>Fisher (Ethicon)</td>
			<td>NC0258473</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube (Opthalamic Ointment/Lubricant)</td>
			<td>Fisher (Henry Schein)</td>
			<td>008897</td>
			<td></td>
		</tr>
	</tbody>
</table>
<table>
	<tbody>
		<tr>
			<td>Table 2. List of Surgical Instruments</td>
		</tr>
		<tr>
			<td>2x Microforceps</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#160-57</td>
			<td>Multi-use (e.g. clearing debris in skull window)</td>
		</tr>
		<tr>
			<td>2x Forceps</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#160-93</td>
			<td>Multi-use (e.g. tying sutures)</td>
		</tr>
		<tr>
			<td>6x Hemostats</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#105-1125</td>
			<td>Clamp and open incision</td>
		</tr>
		<tr>
			<td>1x Small scissors</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#105-411</td>
			<td>Cut sutures after tying</td>
		</tr>
		<tr>
			<td>1x Tissue forceps</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#105-222</td>
			<td>Holding tissue while suturing</td>
		</tr>
		<tr>
			<td>1x Needle holder</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#105-1259</td>
			<td>Holding suture needle</td>
		</tr>
		<tr>
			<td>1x Scalpel holder (with #11 blade)</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#105-80 (w/ #105-71 blade)</td>
			<td>Making skull incision</td>
		</tr>
		<tr>
			<td>1x # 22 Scalpel blade</td>
			<td>George Tiemann &#38; Co.</td>
			<td># 160-381</td>
			<td>Shaving scalp</td>
		</tr>
		<tr>
			<td>1x Surgical Spatula</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#160-718</td>
			<td>Scraping skull to clear tissue on skull</td>
		</tr>
		<tr>
			<td>Machine/Jewelers Screws</td>
			<td>Various</td>
			<td>N/A</td>
			<td>0/80 x 1/8&#8221;</td>
		</tr>
	</tbody>
</table>
<table>
	<tbody>
		<tr>
			<td>Table 3. List of Equipment for Recording Electrophysiological Signals</td>
		</tr>
		<tr>
			<td>Microwire Array &#38; Connector</td>
			<td>Micro Probe, Inc. (Gaithersburg, MD)</td>
			<td>&#160;N/A</td>
			<td>Cranially implanted in target recording region. Arrays are customized based on desired wire spacing, length, etc.</td>
		</tr>
		<tr>
			<td>(Part No. Based on array characteristics)</td>
		</tr>
		<tr>
			<td>Unity-Gain Harness/Headstage</td>
			<td>M.B. Turnkey Designs (Hillsborough, NJ)</td>
			<td>Proj 1200</td>
			<td>Initial amplification of neural signal; allows for propagation of small neural signals.</td>
		</tr>
		<tr>
			<td>Commutator (&#38; Optional Fluid Swivel)</td>
			<td>Plastics One, Inc. (Roanoke, VA)</td>
			<td>SL18C</td>
			<td>Allows animals to freely rotate while propagating electrical signal to preamp</td>
		</tr>
		<tr>
			<td>Pre-Amplifier</td>
			<td>M.B. Turnkey Designs (Hillsborough, NJ)</td>
			<td>Proj 1198</td>
			<td>Differentially amplifies neural signals against a reference electrode.</td>
		</tr>
		<tr>
			<td>Filter &#38; Amplifier</td>
			<td>M.B. Turnkey Designs (Hillsborough, NJ)</td>
			<td>Proj 1199</td>
			<td>Band-pass filters and further amplifies the differentially amplified signal.</td>
		</tr>
		<tr>
			<td>Acquisition Computer</td>
			<td>EnGen (Phoenix, AZ)</td>
			<td>N/A (Custom Build)</td>
			<td>Runs software and hardware for behavioral and neural data acquisition.</td>
		</tr>
		<tr>
			<td>A/D Card&#160;</td>
			<td>Data Translation (Marlboro, MA)</td>
			<td>DT-3010</td>
			<td>Digitizes neural signals for computer sampling.</td>
		</tr>
		<tr>
			<td>Digital I/O Card</td>
			<td>Measurement Computing (Norton, MA)</td>
			<td>PCI CTR-05</td>
			<td>Acquires behavioral inputs and outputs</td>
		</tr>
	</tbody>
</table>

a procedure for implanting organized arrays of microwires for single-unit recordings in awake, behaving animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

A list of Equipment used by this laboratory for recording electrophysiological signals can be found in Table 3. Following recovery from surgery, single-units are recorded by plugging a unity-gain headstage into the implanted connector. This headstage is connected via a cable to a commutator, which is capable of free rotation without breaks in the electrophysiological recording through the use of electrical slip rings. The commutator allows subjects to freely move while recording during behavior, which is...

Watch this Scientific Journal Video about A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals at JoVE.com

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

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