We are grateful to the Canadian Institutes of Health Research (grant MT-5877), the National Institutes of Health (grants NS058668 and NS066664), and the US-Israeli Binational Foundation (grant 2003222) for funding these studies.

Experimental protocols were carried out on female Long Evans rats (250 - 350 g) in accordance with federally prescribed animal care and use guidelines and were approved by the Institutional Animal Care and Use Committee at the University of California San Diego.
1. Acclimating the Rat to Body Restraint
NOTE: Place the rat on a restricted diet. Feed the rat once per day immediately after each daily handling session to acclimate the rat to the restraint (described below...

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Construction of the experimental jig
The description of the mechanical parts used to build the experimental jig (Figure 4) is omitted from the protocol, as it can be constructed in a variety of ways. In this demonstration standard opto-mechanical parts and support clamps are used to mount the head restraint bar and the body restraint tube (see Materials section). Similar opto-mechanical parts can be used to mount the electrode ho...

Monitoring neuronal activity in an alert animal actively engaged in a behavioral task is critical for understanding the function and organization of the nervous system. Extracellular recording of the electrical activity from single neuronal units has long been a staple tool of systems neuroscience and is still widely in use at present. A variety of electrode types and configurations are available depending on the scientific and technical demands of a particular experiment. Chronically implanted microdrives or electrode arrays are often used in freely moving animals, including birds, rodents, and non-human primates1-4. Alternatively, acute penetrations with metal or glass microelectrodes via an external micromanipulator are often used to record from anesthetized or head-restrained animals. Glass micropipette electrodes have the advantage that they can be used in the juxtacellular or &#8220;cell attached&#8221; configuration to unambiguously isolate the activity of single neurons without the complications of post-hoc spike sorting5. These electrodes further permit recording from anatomically-identified cells or locations, as they can be used to inject small deposits of dye or neuroanatomical tracers, or even to fill the individual recorded cell. This configuration has been successfully applied in rats, mice and birds6-10. The presently described technique focuses on juxtacellular monitoring and extracellular dye deposits in alert, head-restrained rats. Note that unlike single cell juxtacellular fills, these dye deposits do not provide information about cell morphology or axonal projections11, but they enable exact anatomical localization to approximately 50 &#956;m and, critically, have a significantly higher yield in alert animals. Information regarding single-cell juxtacellular fills is nonetheless provided as an alternative strategy for anatomical labeling.
In brief, the protocol consists of three major phases. In the first phase, the rat is acclimated to body restraint in a cloth sock (Figure 1) over a period of 6 days. In the second phase, a head restraint apparatus (Figure 2) and recording chamber are surgically implanted such that the rat can be maintained in the stereotaxic plane during multiple subsequent recording sessions (Figure 3); this procedure enables the experimenter to target particular sub-cortical regions of the brain for electrophysiological study based on standard reference coordinates12. The third phase involves placing the rat in an appropriate jig for conducting the behavioral and electrophysiological experiments (Figure 4), constructing the electrode from a quartz capillary tube (Figure 5), making juxtacellular neuronal recordings that unambiguously isolate single units6-9, and marking the anatomical location of the recording site with Chicago Sky Blue dye (Figures 6 and 7). The recordings are performed with simultaneous behavioral monitoring; however, the technical details of the behavior will depend on the scientific goals of each experiment and are thus beyond the scope of a single protocol. After completion of the experimental procedure, which can be repeated on multiple days, the animal is euthanized. The brain is extracted and processed according to standard neuroanatomical techniques using either bright field or fluorescence microscopy.

<table border="0" cellpadding="0" cellspacing="0" width="860">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Name of the Reagent</td>
			<td style="width:179px;">Company</td>
			<td style="width:175px;">Catalogue Number</td>
			<td style="width:315px;">Comments&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Ketaset (Ketamine HCl)</td>
			<td style="width:179px;">Fort Dodge&#160;</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Anased (Xylazine solution)</td>
			<td style="width:179px;">Lloyd Laboratories</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Betadyne (Povidone-Iodine)</td>
			<td style="width:179px;">CVS Pharmacy</td>
			<td align="right" style="width:175px;">269281</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Loctite 495</td>
			<td style="width:179px;">Grainger Industrial Supply</td>
			<td style="width:175px;">4KL86</td>
			<td style="width:315px;">20-40 cp cyanoacrylate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Vetbond</td>
			<td style="width:179px;">3M</td>
			<td style="width:175px;">1469SB</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Grip cement powder</td>
			<td style="width:179px;">Dentsply Intl</td>
			<td align="right" style="width:175px;">675571</td>
			<td style="width:315px;">For the base of the recording chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Grip cement liquid</td>
			<td style="width:179px;">Dentsply Intl</td>
			<td align="right" style="width:175px;">675572</td>
			<td style="width:315px;">For the base of the recording chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Silicone Gel</td>
			<td style="width:179px;">Dow Corning</td>
			<td align="right" style="width:175px;">Mar-80</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Jet denture repair acrylic powder</td>
			<td style="width:179px;">Lang Dental Manufacturing Co.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">For securing the head restraint apparatus to the cranium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:191px;">Ortho-Jet Fast curing orthodontic acrylic resin liquid</td>
			<td style="width:179px;">Lang Dental Manufacturing Co.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">For securing the head restraint apparatus to the cranium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Chicago sky blue</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">C8679</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Paraformaldehyde</td>
			<td style="width:179px;">Sigma</td>
			<td align="right" style="width:175px;">158127</td>
			<td style="width:315px;">For perfusion and tissue fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Phosphate-buffered saline</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">P3813</td>
			<td style="width:315px;">For perfusion and tissue fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Cytochrome C</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">C2506</td>
			<td style="width:315px;">For cytochrome-oxidase staining (Figure 7)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Diaminobenzidine</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">D5905</td>
			<td style="width:315px;">For cytochrome-oxidase staining (Figure 7)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Material Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:175px;">Catalogue Number</td>
			<td style="width:315px;">Comments&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Rat sock</td>
			<td style="width:179px;">Sew Elegant (San Diego, CA)</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">Custom made, Figures 1, 4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">PVC tube 2 &#189;&#8221;</td>
			<td style="width:179px;">U.S. Plastic Co.</td>
			<td align="right" style="width:175px;">34108</td>
			<td style="width:315px;">Figure 4</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Subminiature D pins &#38; sockets</td>
			<td style="width:179px;">TE Connectivity</td>
			<td style="width:175px;">205089-1</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Stainless steel music wire 0.010&#8221; diameter</td>
			<td style="width:179px;">Precision Brand Products, Inc.</td>
			<td align="right" style="width:175px;">21010</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Stereotaxic holding frame</td>
			<td style="width:179px;">Kopf Instruments</td>
			<td style="width:175px;">Model 900</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Stereotaxic ear bars</td>
			<td style="width:179px;">Kopf Instruments</td>
			<td style="width:175px;">Model 957</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Stereotaxic manipulator</td>
			<td style="width:179px;">Kopf Instruments</td>
			<td style="width:175px;">Model 960</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">&#189; mm drill burr</td>
			<td style="width:179px;">Henry Schein</td>
			<td style="width:175px;">100-3995</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Quiet-Air dental drill&#160;</td>
			<td style="width:179px;">Midwest Dental</td>
			<td style="width:175px;">393-1600</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Stainless steel 0-80 1/8&#8221; screw</td>
			<td style="width:179px;">Fastener superstore</td>
			<td align="right" style="width:175px;">247438</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">0.2mL centrifuge tube</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:175px;">05-407-8A</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Custom head-holding bar</td>
			<td style="width:179px;">UCSD SIO Machine Shop</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">Custom made, Figures 2, 3, 4</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:191px;">Custom head-holding plate</td>
			<td rowspan="2" style="width:179px;">UCSD SIO Machine Shop</td>
			<td rowspan="2" style="width:175px;">N/A</td>
			<td rowspan="2" style="width:315px;">Custom made, Figure 2, 3, 4</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:191px;">Right angle post-clamp</td>
			<td style="width:179px;">Newport</td>
			<td style="width:175px;">MCA-1</td>
			<td style="width:315px;">Figure 3,4; standard opto-mechanical parts for the experimental jig (Figure 4) are also from Newport Corp.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">8-32 3/4&#8221; screw</td>
			<td style="width:179px;">Fastener Superstore</td>
			<td align="right" style="width:175px;">240181</td>
			<td style="width:315px;">For head-restraint, Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">4-40 &#188;&#8221; screw</td>
			<td style="width:179px;">Fastener Superstore</td>
			<td align="right" style="width:175px;">239958</td>
			<td style="width:315px;">For head restraint, Figures 3, 4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Quartz capillary tubing</td>
			<td style="width:179px;">Sutter Instruments</td>
			<td style="width:175px;">QF-100-60-10</td>
			<td style="width:315px;">Figure 5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Carbon dioxide laser puller</td>
			<td style="width:179px;">Sutter instruments</td>
			<td style="width:175px;">P-2000</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Motorized micromanipulator</td>
			<td style="width:179px;">Sutter Instruments</td>
			<td style="width:175px;">MP-285</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Microelectrode amplifier</td>
			<td style="width:179px;">Molecular Devices</td>
			<td style="width:175px;">Multiclamp 700B</td>
			<td style="width:315px;">Alternate part: Molecular Devices Axoclamp 900A</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Microelectrode amplifier head stage</td>
			<td style="width:179px;">Molecular Devices</td>
			<td style="width:175px;">CV-7B</td>
			<td style="width:315px;">Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Isolated pulse stimulator</td>
			<td style="width:179px;">A-M Systems</td>
			<td style="width:175px;">Model 2100</td>
			<td style="width:315px;">Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Audio monitor</td>
			<td style="width:179px;">Radio Shack</td>
			<td style="width:175px;">32-2040</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:191px;">Pipette holder</td>
			<td rowspan="2" style="width:179px;">Warner Instruments</td>
			<td rowspan="2" style="width:175px;">#MEW-F10T</td>
			<td style="width:315px;">Alternate parts: see Discussion</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Figure 6A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Electrode lead wire</td>
			<td style="width:179px;">Cooner wire</td>
			<td style="width:175px;">NEF34-1646</td>
			<td style="width:315px;">(optional), Figure 6D</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:191px;">Relay for amplifier head-stage</td>
			<td style="width:179px;">COTO Technology</td>
			<td style="width:175px;">#2342-05-000</td>
			<td style="width:315px;">(optional) Used with a custom-made printed circuit board (UCSD Physics Electronics Shop), Figure 6A-C</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Digital video camera</td>
			<td style="width:179px;">Basler</td>
			<td style="width:175px;">A602fm</td>
			<td style="width:315px;">(optional) For behavioral monitoring, Figure 7</td>
		</tr>
	</tbody>
</table>

juxtacellular monitoring and localization of single neurons within sub-cortical brain structures of alert, head-restrained rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

Neuronal units in ventral posterior medial (VPM) thalamus encode the phase of vibrissa movement during self-generated whisking15,16. Figure 7A shows sample spiking activity of a VPM thalamic unit as a rat is actively whisking. Figure 7B shows a histogram of spike times aligned to the instantaneous phase of vibrissa motion17. There are more spikes during the retraction phase of whisking. After the recording, the location of the unit was labeled via iontophoresis of C...

Watch this Scientific Journal Video about Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats at JoVE.com

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

juxtacellular-monitoring-localization-single-neurons-within-sub

Research

JoVE Journal

Neuroscience

11.2K vues.  University of California, San Diego. This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.