The work was supported by R01 DC 011582 (AK) and RO1 DC011555 (DJT).

1. Pull Glass Electrodes
<ol>
	<li>Pull single barrel electrode. Use single barrel glass capillary with filament and pull tip to a diameter of about 1-2 micrometers, shaft length of 10-12 mm, and corresponding electrode resistance of about 12 MOhm (range 5-20 MOhms) as measured in 0.9% NaCl solution. Lower electrode resistances would result in more background activity and thus more difficulty in isolating unitary activity from individual neurons. For pulling this electrode, use either a horizontal or a vertical puller with either heating filaments or heating coils.</li>
	<li>Pull multibarrel electrode. Due to the much larger diameter of multibarrel glass and the need of even heat distribution around the entire multibarrel, a powerful puller with a larger diameter heating coil, not a heating filament, is needed. A multibarrel pipette has to be inserted in the center of the heating filament with no contact to the heating coil. Note that besides the 5-barrel pipettes described here, 3-barrel or 7-barrel pipettes are commercially available alternatives. Pull glass to a pipette tip of about 10 micrometers total diameter, or less. The tip will be broken to the correct diameter in the next step, so the exact tip size is less critical during the pulling process than the overall shape of the electrode tip, which should be long and relatively thin. Refer to the image in Figure 1C for the desired electrode tip shape. Short and stubby electrode shapes (Figure 1B) will cause a substantial amount of tissue damage when advanced into the brain, while very long and thin electrode shapes (Figure 1A) will bend and thus will make it difficult to break the electrode tip to the correct diameter (see step 2).</li>
</ol>
2. Modify Electrode Tips
Before the two electrodes can be glued together, they have to be modified. The shaft of the single electrode needs to be bent before it can be attached to the multibarrel to make sure the combined shaft of the finished piggy-back electrode is as thin as possible. Additionally, the tip of the multibarrel electrode has to be broken off in order to ensure low resistance for iontophoresis.
<ol>
	<li>Bend shaft of a single barrel electrode by about 20 degrees. Use the smallest Bunsen burner flame possible. Typical "small" Bunsen burners from standard lab supply companies create flame sizes that are far too large for this application. To circumvent this problem use the smallest commercial Bunsen burner and secure a syringe needle (~ 18 gauge) to the top of the burner, and seal the connection using dental cement. When operated, the flame should be difficult to see, about 5 mm or less in diameter, and about 8 mm tall. Any air movement in the room will extinguish that flame, so it is a good idea to operate the burner in a closed room, or to use wind shields. Move the single barrel electrode through the flame to bend it by about 20 degrees. Aim to have the burner flame melt the glass at the transition area at about 10 millimeters away from the electrode tip. To avoid melting the tip of the electrode we suggest that the electrode be held at about 45 degrees, tip pointing downward, and move the electrode through the flame relatively quickly.</li>
	<li>Break off the tip of the multibarrel electrode. To ensure visual control while breaking the electrode tip, use a microscope with a minimum 10x objective and 10x oculars. A measuring scale inserted in the ocular will also be needed to measure tip sizes. Attach a piece of plexiglass to the microscope such that the end of the plexiglass can be seen in about one-third to one-half of the field of view of the microscope. In our case, the plexiglass piece is about 25 x 70 mm and 5 mm thick and attached via a screw that can be secured into a custom made thread in the microscope stage. It is important to have a design that allows for the plexiglass moving independently of the slide. Place the multibarrel electrode in a bed of modeling clay on a glass slide, and insert the slide containing the electrode into the microscope stage's slide holder. Using the microscope stage's xy manipulators, gently move the electrode tip against the plexiglass piece, and observe the breaking of the tip through the microscope oculars. Attempt to cleanly break the multibarrel's tip to a cumulative diameter of about 25-35 micrometers. Discard pipettes with tip diameters that broke off too large, or tips with uneven breaks. We discard approximately 30% of our multibarrel electrodes due to undesirable tip shapes.</li>
</ol>
3. Assemble Piggy-back Electrode
<ol>
	<li>Position electrodes. Remove plexiglass piece used in step 2.2 from the microscope stage. Secure completed multibarrel electrode into modeling clay on a glass slide, tip pointing slightly upward. Pointing upward will be important for step 3.2, the gluing of the two electrodes. Tips that point up cause the glue to run away from the tip, avoiding gluing the electrode tips. Insert bent single barrel electrode into electrode holder of the custom made micromanipulator (Figure 2). Using visual guidance first and then microscopic guidance, lower the single barrel electrode onto the multibarrel electrode. The single electrode should be lowered directly into the groove that is formed by the arrangement of the 5 barrels, with its tip protruding the tip of the multibarrel tip by about 5-10 micrometers. When lowering the single barrel, closely observe the angle that is formed between the two electrodes. For best outcome avoid any angles in which the tips point apart from each other, but rather attempt to lower the single electrode onto the multi perfectly parallel or even with its tip touching the multibarrel first, forming a VERY slight 'wedge' arrangement. Since the single barrel tip is very flexible, it will bend when the single electrode is lowered a little further after the tip has reached the top surface of the multi barrel electrode, forming a clean composite tip that has a small amount of spring action built in that helps holding the tips together. However, if the angle between single barrel and multibarrel electrode is too steep (too much of a wedge), the spring action will be too high and bend the electrode arrangement downwards.</li>
	<li>Glue electrode shafts together. Glue the shafts of the two electrodes together using cyanoacrylate (superglue). Place a small drop of glue onto the small side of a flat toothpick and touch electrode assembly with the glue drop. Start at the position most distal of the tips and slowly move toothpick with glue drop along electrode shafts towards the electrode tips. Using too much glue, or applying glue too close to the electrode tips will result in gluing the electrode openings, rendering the electrode at least partially nonfunctional.</li>
	<li>Stabilize joint with dental cement. Mix a small amount of dental cement and dental acrylic in a small disposable plastic dish or weigh boat, using a flat toothpick. Wait until cement becomes moldable and apply a small amount to the joint between the two electrodes to stabilize the joint (pink material in Figure 3). Allow about 15 min to dry.</li>
	<li>Remove and store electrode. Carefully remove the completed piggy-back electrode first from the micromanipulator holder, and then detach from the glass slide, and store in a dustproof container.</li>
</ol>
4. Prepare Electrode Fill Solutions
Prepare electrode fill solutions. Since iontophoresis requires charged molecules, most agents have to be dissolved either in an acidic or alkaline environment (typically at a pH of about 3-4, or a pH of about 8-10, respectively). A number of chemicals that are often used in iontophoresis are listed in Table 1. For agents that are not listed in the table, determine from the pKa value, whether it would be easier to use the molecule in an acidic or an alkaline environment to keep the molecule charged, and dissolve accordingly. For best results, mix all solutions fresh daily.
5. Fill and Prepare Electrodes
Just before using the electrode, back-fill each barrel with its respective drug, using carbon fiber 28 - 34 gauge needles attached to syringes with syringe filters. Fill the 4 outer barrels of the 5-barrel configuration with the drugs of choice, and the center barrel with 3M NaCl as a balancing barrel. Fill the single barrel recording electrode with 3M NaCl as well. Adding a dye to the NaCl solution, such as fast green or phenol red will make it easier to see the electrode tip during placement of the electrode onto the brain surface. Insert the electrode into the electrode holder of the recording setup and insert all wires into the appropriate glass barrels. Use insulated silver wire from which about 1 cm of insulation has been removed at the tip. There should be 5 wires for the multibarrel electrode (4 drug barrels and one balancing barrel), plus the amplifier wire that needs to be inserted into the recording single barrel electrode.
6. Turn on Iontophoresis Pump Modules
Turn on iontophoresis pump modules and test all the barrels. The electrode test function of each pump module will help determine if the electrode barrel is functional. To prevent leakage of the drugs from the barrels when not in use, a retention voltage in the opposite polarity as the molecule charge needs to be applied.

<ol>
	<li>Curtis, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+assembly+of+"parallel"+micro-pipettes.">A method for assembly of "parallel" micro-pipettes.</a> Electroencephalogr. Clin. Neurophysiol. 24, 587-589 (1968).</li><li>Carette, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+manufacturing+multi-barrelled+micropipettes+with+projecting+recording+barrel.">A new method of manufacturing multi-barrelled micropipettes with projecting recording barrel.</a> Electroencephalogr. Clin. Neurophysiol. 44, 248-250 (1978).</li><li>Crossman, A. R., Walker, R. J., Woodruff, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Problems+associated+with+iontophoretic+studies+in+the+caudate+nucleus+and+substantia+nigra.">Problems associated with iontophoretic studies in the caudate nucleus and substantia nigra.</a> Neuropharmacology. 13, 547-552 (1974).</li><li>Havey, D. C., Caspary, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+constructing+"piggy-back"+multibarrel+microelectrodes.">A simple technique for constructing "piggy-back" multibarrel microelectrodes.</a> Electroencephalogr. Clin. Neurophysiol. 48, 249-251 (1980).</li><li>Verberne, A. J., Owens, N. C., Jackman, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+reliable+method+for+construction+of+parallel+multibarrel+microelectrodes.">A simple and reliable method for construction of parallel multibarrel microelectrodes.</a> Brain Res. Bull. 36, 107-108 (1995).</li><li>Oliver, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+contribution.+A+simple+rapid+method+for+preparing+parallel+micropipette+electrodes.">Technical contribution. A simple rapid method for preparing parallel micropipette electrodes.</a> Electroencephalogr. Clin. Neurophysiol. 31, 284-286 (1971).</li><li>Oswald, J. P., Klug, A., Park, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaural+intensity+difference+processing+in+auditory+midbrain+neurons:+effects+of+a+transient+early+inhibitory+input.">Interaural intensity difference processing in auditory midbrain neurons: effects of a transient early inhibitory input.</a> J. Neurosci. 19, 1149-1163 (1999).</li><li>Park, T. J., Pollak, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+shapes+a+topographic+organization+of+response+latency+in+the+mustache+bat's+inferior+colliculus.">GABA shapes a topographic organization of response latency in the mustache bat's inferior colliculus.</a> J. Neurosci. 13, 5172-5187 (1993).</li><li>Park, T. J., Pollak, G. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT1A+and+5-HT1B+receptors+differentially+modulate+rate+and+timing+of+auditory+responses+in+the+mouse+inferior+colliculus.">5-HT1A and 5-HT1B receptors differentially modulate rate and timing of auditory responses in the mouse inferior colliculus.</a> Eur. J. Neurosci. 32, 368-379 (2010).</li><li>Wenstrup, J. J., Leroy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+Integration+in+the+Inferior+Colliculus:+Role+of+Glycinergic+Inhibition+in+Response+Facilitation.">Spectral Integration in the Inferior Colliculus: Role of Glycinergic Inhibition in Response Facilitation.</a> J. Neurosci. 21, RC124 (2001).</li><li>Yang, L., Pollak, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+ipsilaterally+evoked+inhibition+in+the+dorsal+nucleus+of+the+lateral+lemniscus.">Features of ipsilaterally evoked inhibition in the dorsal nucleus of the lateral lemniscus.</a> Hear. Res. 122, 125-141 (1998).</li><li>Yang, L., Pollak, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+and+glycine+have+different+effects+on+monaural+response+properties+in+the+dorsal+nucleus+of+the+lateral+lemniscus+of+the+mustache+bat.">GABA and glycine have different effects on monaural response properties in the dorsal nucleus of the lateral lemniscus of the mustache bat.</a> J. Neurophysiol. 71, 2014-2024 (1994).</li><li>Yang, L., Pollak, G. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+inhibition+in+creating+complex+auditory+responses+in+the+inferior+colliculus:+facilitated+combination-sensitive+neurons.">Roles of inhibition in creating complex auditory responses in the inferior colliculus: facilitated combination-sensitive neurons.</a> J. Neurophysiol. 93, 3294-3312 (2005).</li><li>Fukui, I., Burger, R. M., Ohmori, H., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+inhibition+sharpens+the+frequency+tuning+and+enhances+phase+locking+in+chicken+nucleus+magnocellularis+neurons.">GABAergic inhibition sharpens the frequency tuning and enhances phase locking in chicken nucleus magnocellularis neurons.</a> J. Neurosci. 30, 12075-12083 (2010).</li><li>Burger, R., Pollak, G. 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Neurophysiol. 80, 1686-1701 (1998).</li><li>Coleman, W. L., Fischl, M. J., Weimann, S. R., Burger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+and+glycinergic+inhibition+modulate+monaural+auditory+response+properties+in+the+avian+superior+olivary+nucleus.">GABAergic and glycinergic inhibition modulate monaural auditory response properties in the avian superior olivary nucleus.</a> J. Neurophysiol. 105, 2405-2420 (2011).</li></ol>

No conflicts of interest declared.

We describe a technique that allows for the manipulation of a single neuron's microcircuit in vivo, while at the same time allowing for the recording of the neuron's responses during the experimental manipulation. Neural circuits are manipulated via the iontophoretical application of synaptic agonists and antagonists. The main advantage of iontophoresis over pressure ejection is that iontophoresis does not require the physical movement of fluid from the electrode into neural tissue, and thus there is no concern of causing tissue damage through the applied pressure or fluid volume. The major limitation of this technique is lack of information about the absolute drug concentration in the tissue, and the volume of tissue affected. However, since the amounts of pharmacological agents ejected with iontophoresis are much smaller and much more precisely controllable than with pressure ejection, the recovery from the drug application is typically much faster and much more complete. Microiontophoresis has successfully been used in a number of neural systems, sensory and others, and is applied most successfully in brain areas with little or no intrinsic processing. The reason is that some of the ejected pharmacological agent may diffuse from the application site to a neighboring neuron and also manipulate the response properties of the neighboring neuron.
The separate manufacturing of single and multi barrel electrodes allows for the combination of electrodes with arbitrary and unrelated properties. Pulling electrode barrels together and using some for recording and some for iontophoresis purposes would produce electrode tips with very similar properties, such that the electrode tips would either be too large for single cell recording, or too small for drug application. Also, having the single barrel tip extend beyond the multibarrel electrode tips by about 20 micrometers greatly reduces noise in the recordings, and eliminates possible confounding current effects from the retention or ejection currents on the neuron's firing 3.
Piggy-back multibarrel electrodes have first been described over 30 years ago 4-6 and have been used very successfully to dissect neural circuits 7-18 19-29.Thus, the method per se is not novel or unique . However, the particular details of electrode preparation and use have been modified over the years, and the set of instructions described here has proven to be especially easy and successful, and has not been published in detail elsewhere in the literature. Particularly, the bending of the single barrel electrode tip allows the final tip of the piggy-back electrode to be relatively slim (Figure 3) and thus, allows for recordings from deep nuclei with minimal damage to the brain; the protruding of the single barrel electrode over the multi-barrel electrode removes virtually all current effects, which were often cited as a disadvantage of the technique 3. New details presented here such as having the electrode tip pointing upward during the gluing process and resting the single barrel in the groove of the multibarrel electrode will ensure a high success rate when producing piggy-back electrodes. The technique is relatively easy and can typically be mastered by a novice within a few days.

<table border="1">
	<tbody>
		<tr>
			<td>Item name</td>
			<td>Manufacturer</td>
			<td>Comment</td>
			<td>Cat. #</td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>&nbsp;</td>
			<td>Available from: VWR</td>
			<td>17928-027</td>
		</tr>
		<tr>
			<td>Two-component dental cement: "Cold cure" dental material</td>
			<td>Co-oral-ite Dental Mfg. Co</td>
			<td>Available from: A-M Systems, Inc</td>
			<td>525000</td>
		</tr>
		<tr>
			<td>Two-component dental cement: Denture material crosslinking Liquid Compound</td>
			<td>Co-oral-ite Dental Mfg. Co</td>
			<td>Available from: A-M Systems, Inc</td>
			<td>525000</td>
		</tr>
		<tr>
			<td>Liquid glue</td>
			<td>Henkel</td>
			<td>Available from: Loctite Super Glue</td>
			<td>01-06849</td>
		</tr>
		<tr>
			<td>Micro-Iontophoresis Unit: Neurophore BH-2</td>
			<td>Harvard Apparatus</td>
			<td>Available from: Harvard Apparatus</td>
			<td>65-0200 &amp; 65-0203</td>
		</tr>
		<tr>
			<td>Insulated silver wire</td>
			<td>AM-Systems</td>
			<td>Available from: AM-Systems</td>
			<td>785500</td>
		</tr>
		<tr>
			<td>Horizontal puller</td>
			<td>Zeitz DMZ-Universal Puller</td>
			<td>Available from: AutoMate Scientific</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Micro-manipulator pieces: electrode holder</td>
			<td>WPI</td>
			<td>Available from: WPI</td>
			<td>M3301EH</td>
		</tr>
		<tr>
			<td>Micro-manipulator pieces: linear stage</td>
			<td>Newport 423 Series</td>
			<td>Available from: Newport</td>
			<td>423</td>
		</tr>
		<tr>
			<td>Micro-manipulator pieces: rotation stage</td>
			<td>Newport RSP-2</td>
			<td>Available from: Newport</td>
			<td>RSP-2</td>
		</tr>
		<tr>
			<td>Micro-manipulator pieces: z translation</td>
			<td>Newport 433 Series</td>
			<td>Available from: Newport</td>
			<td>433</td>
		</tr>
		<tr>
			<td>Micro-manipulator pieces: angle bracket 90 &deg; to assemble z and xy axis</td>
			<td>Newport 360-90</td>
			<td>Available from: Newport</td>
			<td>360-90</td>
		</tr>
		<tr>
			<td>Micro-manipulator pieces: x translation / linear stage</td>
			<td>Newport 423 Series</td>
			<td>Available from: Newport</td>
			<td>423</td>
		</tr>
		<tr>
			<td>Micro-manipulator pieces: y translation / linear stage</td>
			<td>Newport 423</td>
			<td>Available from: Series Newport</td>
			<td>423</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leitz Laborlux 11</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope: objective</td>
			<td>Leitz Wetzlar 10x, NA 0.25</td>
			<td>&nbsp;</td>
			<td>519760</td>
		</tr>
		<tr>
			<td>Microscope: eypieces</td>
			<td>Leitz Wetzlar, Periplan 10x/18</td>
			<td>&nbsp;</td>
			<td>519748</td>
		</tr>
		<tr>
			<td>Microscope: stage</td>
			<td>Leitz Wetzlar</td>
			<td>&nbsp;</td>
			<td>513544</td>
		</tr>
		<tr>
			<td>Multibarrel capillary</td>
			<td>N/A</td>
			<td>Available from: A-M systems, Inc</td>
			<td>612000</td>
		</tr>
		<tr>
			<td>Sinlge barrel capillary (GC 150F-10)</td>
			<td>Harvard Apparatus</td>
			<td>Available from: Harvard Apparatus</td>
			<td>30-0057</td>
		</tr>
		<tr>
			<td>Vertical puller</td>
			<td>Narishige model PE-2</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Custom made elements of the Micro-manipulator (marked light blue in Figure 1)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>steel plate</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>tilting base</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>attachment for electrode holder</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>
			Table 2. Manufacturers and item numbers of all equipment and supplies used in the procedure.</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

manufacturing and using piggy-back multibarrel electrodes for in vivo pharmacological manipulations of neural responses

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neuron's output caused by this manipulation. This can be accomplished by pharmacologically altering the neuron's immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function.
Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago 1-3, and have undergone a number of design improvements 2,3 until the piggy-back type was introduced in the 1980s 4,5. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.

In this experiment, the glycine receptor antagonist strychnine hydrochloride was iontophoretically applied. Blocking glycinergic inhibition typically increases firing in neurons. Figure 4 shows sample data from an auditory neuron whose responses to sinusoidal sound stimuli of increasing intensity delivered to the animal's ears were recorded. This type of an experiment is referred to as the neuron's discharge rate vs intensity function. Louder sounds resulted in higher spike rates (black curve). The initial iontophoresis current used during this experiment was 15 nA. After the current was switched on and the changes in the rate intensity function had stabilized at their new level (dark blue curve), the ejection current was progressively increased to 30, 45, and 60 nA (orange, green, and light blue curves, respectively). In each case, the responses of the neuron over the same range of sound intensities were recorded after the changes in the discharge rate-intensity functions in response to the new ejection current had stabilized. The most appropriate ejection current to use in this example was 45 nA to 60 nA because these levels of current no longer alter differently the neuron's responses. This result suggests that at 45 nA current, all glycine receptors of that neuron had already been blocked by strychnine hydrochloride. Any further increase of the ejection current and releasing even more strychnine did not result in a further change of the neuron's discharge rate-level function. After the completion of the protocol, the ejection current was turned off. The recovery of neural responses back to baseline was achieved after about 25 min (red line). This might take, depending on the type and amount of drug ejected, between several seconds and several tens of minutes.
<table border="1">
	<tbody>
		<tr>
			<td>Drug</td>
			<td>Concentration</td>
			<td>pH of a solution</td>
			<td>Solvent</td>
			<td>Company</td>
			<td>Cat. #</td>
			<td>Typical Retention Current</td>
			<td>Typical Ejection Currents</td>
		</tr>
		<tr>
			<td>GABA</td>
			<td>500 mM</td>
			<td>3.5-4.0</td>
			<td>dH2O</td>
			<td>Sigma</td>
			<td>A-2129</td>
			<td>-15 nA</td>
			<td>+5 nA to +100 nA</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>100 mM</td>
			<td>3.5-4.0</td>
			<td>dH2O</td>
			<td>Sigma</td>
			<td>G-7126</td>
			<td>-15 nA</td>
			<td>+5 nA to +100 nA</td>
		</tr>
		<tr>
			<td>Bicuculline Methiodide</td>
			<td>10 mM</td>
			<td>3.0</td>
			<td>0.165 M NaCl in dH2O</td>
			<td>Sigma</td>
			<td>B-6889</td>
			<td>-15 nA</td>
			<td>+5 nA to +60 nA</td>
		</tr>
		<tr>
			<td>Strychnine hydrochloride</td>
			<td>10 mM</td>
			<td>3.0</td>
			<td>0.165 M NaCl in dH2O</td>
			<td>Sigma</td>
			<td>S-8753</td>
			<td>-15 nA</td>
			<td>+5 nA to +80 nA</td>
		</tr>
		<tr>
			<td>L-Glutamic Acid</td>
			<td>500 mM</td>
			<td>8.0</td>
			<td>dH2O</td>
			<td>Sigma</td>
			<td>G-1251</td>
			<td>+30 nA</td>
			<td>-10 nA to -150 nA</td>
		</tr>
		<tr>
			<td>L-Aspartic Acid</td>
			<td>500 mM</td>
			<td>8.0</td>
			<td>dH2O</td>
			<td>Sigma</td>
			<td>A-8949</td>
			<td>+30 nA</td>
			<td>-10 nA to -150 nA</td>
		</tr>
		<tr>
			<td>Kainic Acid</td>
			<td>1 mM</td>
			<td>9.0</td>
			<td>dH2O</td>
			<td>Sigma</td>
			<td>K-0250</td>
			<td>+30nA</td>
			<td>-10nA to -100 nA</td>
		</tr>
	</tbody>
</table>
Table 1. Commonly used drugs, with pH for dissolving and concentration. The table lists the most commonly used synaptic agonists and antagonists used with iontophoresis. The pH environment listed accounts for the need to polarize these agents, and the suggested concentration accounts for the variability in effectiveness between different drugs.
<img alt="Figure 1" src="/files/ftp_upload/4358/4358fig1.jpg" /> 
Figure 1. Three multibarrel pipettes with different tip lengths. A: The tip of this 5-barrel electrode has been pulled too long and thin. Note that the tip is bent and very soft. This type of tip is very difficult to break to the desired diameter. B: The tip of this electrode is too short and stubby. When advanced into deeper brain areas, this electrode will cause unnecessary brain damage due to the fact that the electrode becomes relatively thick just a few millimeters after the tip. C: An example of an electrode with a correctly pulled tip. While being long and thin, the tip is still firm and can be broken easily to the desired tip diameter.
<img alt="Figure 2" src="/files/ftp_upload/4358/4358fig2.jpg" /> 
Figure 2. Drawing of electrode manipulator assembly. The manipulator assembly is used together with a microscope to assemble the piggy-back electrodes. Items marked in grey are commercially available products and are listed in Table 2. Items marked in blue were custom machined at our institution's machine shop. They are 1) 1/4 inch steel plate sized 43x26 cm with holes for Newport stage 423 drilled into it according to the hole pattern provided by Newport; 2) a tilting stage that allows for tilting of the assembly at arbitrary angles; 3) a connector that mounts the electrode holder to the top translational stage.
<img alt="Figure 3" src="/files/ftp_upload/4358/4358fig3.jpg" /> 
Figure 3. Photo of a sample piggy-back electrode. A finished 5-barrel electrode assembled together with a single-barrel recording electrode. Note long shaft of about 7mm allowing for a deep brain recordings.
<img alt="Figure 4" src="/files/ftp_upload/4358/4358fig4.jpg" /> 
Figure 4. Titration of ejection currents. The graph shows rate-intensity functions recorded from a single auditory neuron while the animal's ears were stimulated with tones of various intensities. Louder sounds tended to elicit higher firing rates. Before drug application, the neuron's rate-intensity function showed the lowest spike rates (black curve). Progressively higher ejection currents blocked progressively more glycine receptors at the neuron, resulting in progressively higher firing rates. The optimal ejection current in this neuron was 45-60 nA. With these ejection currents, complete blockage of all the neuron's glycine receptors was achieved. After completion of the experimental protocol, the iontophoresis was terminated and the neuron was allowed to recover. Complete recovery was achieved when the recovery rate-intensity function matched the initial pre-drug recovery function. Reproduced, with permission from the American Physiological Society, from Klug et al, 1995.

Watch this Scientific Journal Video about Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses at JoVE.com

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron&#x2019;s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. ...

manufacturing-using-piggy-back-multibarrel-electrodes-for-vivo

Research

JoVE Journal

Neuroscience

10.4K Views.  University of Colorado Medical Campus. Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron’s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.

Video: Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...