Name: Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain (Video) | JoVE Journal
Uploaded: 2015-07-07T13:00:00.000+00:00
Duration: 7 min 52 s
Description: Learn to construct a dual-function glass pipette for simultaneous microinjection of GABA and electrophysiological recording in the rat superior colliculus. This affordable method enables real-time monitoring of neuronal activity during targeted drug delivery.

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5 minutes

Supported by grants from CIHR (MOP231122) and NSERC (RGPIN-2014-06503). We would like to thank Genevi&#232;ve Cyr for her help preparing experiments and supervising laboratory work. MAL received a scholarship from The Natural Sciences and Engineering Research Council of Canada (NSERC).

NOTE: All procedures were performed in accordance with the directives of the Canadian Council for the Protection of Animals and the Ethics review board of the Universit&#233; de Montr&#233;al.
1. Assembly of the Recording-injection Pipette
<ol>
	<li>Pull an approximately 7 cm long glass capillary (1 mm outer diameter) using a pipette puller.</li>
	<li>Break the tip of the capillary and check the aperture under a light microscope. Confirm that the inner diameter is between 30 &#181;m to 40 &#181;m.</li>
	<li>Insert a 7 cm long silver wire into the glass capillary with approximately 1 cm protruding from the non-tapered end of the glass pipette.</li>
	<li>Bend the excess filament orthogonally to the glass capillary.</li>
	<li>Apply a droplet of flexible plastic adhesive on the shaft of a 30 G hypodermic needle.</li>
	<li>Insert a 30 G hypodermic needle in the glass pipette according to the schematics presented in Figure 1.</li>
	<li>Add a second coating of glue to ensure a proper seal from the junction between the glass pipette and the hypodermic needle.</li>
	<li>Leave the pipette to dry with the tip facing upward for about 12 hr to ensure proper curing of the glue. The finished result is shown in Figure 2.</li>
</ol>
NOTE: This procedure is done on acute experiments and sterilization of the pipette tip is not required.
2. Animal Preparation
<ol>
	<li>Place the rat in an anesthesia box.</li>
	<li>Induce anesthesia using 4% isoflurane for 5 to 10 min.</li>
	<li>Place the animal on a stereotaxic table with heating pad and rectal probe to maintain a body temperature of 37 &#176;C. Use a nose cone to maintain anesthesia with 2% isoflurane. Secure the rat&#8217;s head using ear bars and teeth holder.</li>
	<li>Apply ophthalmic ointment or eye drops, which include 1% Atropine drops to aid in pupil dilation.&#160; To prevent dryness, apply lubricating drops approximately every 30 min.</li>
	<li>Shave the head and clean it with 10% povidone-Iodine.</li>
	<li>For local anaesthesia, inject 0.5 ml of 2% lidocaine under the scalp in 2-3 locations by lifting up the skin and inserting the tip of the needle.</li>
	<li>Confirm adequate level of anesthesia by performing a toe pinch and observing the lack of movement. In addition, monitor the heart rate to ensure that it is within normal values (300 to 400 beats/min).</li>
	<li>Incise the scalp in a straight line along the median with a #10 scalpel blade to expose both the coronal and sagittal sutures.</li>
	<li>Reveal the Lambda and Bregma points by pushing aside the tissue that is covering the cranium with a surgical spatula.</li>
	<li>Level the cranium so that Bregma and Lambda positions are on the same plane.</li>
	<li>To set the reference point, use a stereotaxic device with a mounted glass tube to set it right above Bregma. This will be the &#8220;zero&#8221; for the antero-posterior and medial-lateral measurements coordinates.</li>
	<li>Set the point of interest by moving the stereotaxic mount to the required coordinates, note the stereotaxic coordinates and draw a square around the target area that marks where the craniotomy will be performed.</li>
	<li>Use a surgical drill with a sterilized drill along the marked square slowly without pressure to slowly remove the bone material. Be careful not to drill too long in the same area, as it will produce heat and cause lesions on the cortex.</li>
	<li>When the bone delimiting the craniotomy has become sufficiently thin, carefully remove the cranial section with tweezers to expose the cortex.</li>
	<li>Frequently irrigate the exposed cortex with artificial cerebral spinal fluid to prevent tissue desiccation. 
	NOTE: Dura mater removal is unnecessary on the rat as the tip of the injectrode is sturdy enough to penetrate.</li>
</ol>
3. Filling and Mounting of the Injection System
<ol>
	<li>Fill the 5-10 &#181;l microsyringe by aspiration with mineral oil.</li>
	<li>Fill the hypodermic needle with the fixed glass pipette with a solution of 0.5% Chicago Sky Blue (CSB) and 300 &#181;M &#947;-aminobutyric acid (GABA) or a solution of 2% lidocaine with 0.5% CSB 9. Dilute all solutions with saline.
	<ol>
		<li>In the case of an abundant substance, fill using a regular syringe and use the usual precautionary techniques to avoid the formation of air bubbles.</li>
		<li>In the case of more expensive substances, use mineral oil to fill the injectrode and the chemical agent can then be introduced by aspiration. As the density difference between mineral oil and water is relatively high, this substance is a good candidate for the injection of aqueous solutions. 
		NOTE: A dye can be added to confirm the separation between both liquids.</li>
	</ol>
	</li>
	<li>To fill the injection pipette, fill a 1 ml syringe with the solution by aspiration and then slowly inject the solution into the injection pipette.</li>
	<li>Pay careful attention for leaks in regions indicated in Figure 1 by swabbing these areas clean and observing leakages by further injecting the solution slowly with the syringe.</li>
	<li>Remove the 1 ml syringe. When doing so, be sure to keep light pressure on the plunger so that the vacuum does not remove the solution from the injection pipette.</li>
	<li>Fill the microsyringe with mineral oil by aspiration and attach it firmly to the filled injection pipette, then carefully wipe away any excess solution from the assembled injectrode with gauze.</li>
	<li>Verify that the tip is not blocked by injecting a very small volume, enough to see a small drop forming at the tip of the glass pipette.</li>
	<li>Mount the injectrode on the micropump system and ensure that it is well fixed.</li>
	<li>Carefully position the injectrode tip at the target coordinates and lower the tip to the surface of the cortex.</li>
	<li>Slowly lower the injectrode using the stereotaxic apparatus to the target structure (superior colliculus in this case) using the appropriate anatomical coordinates.</li>
	<li>Cover the exposed cortex with warm agar to prevent tissue desiccation.</li>
</ol>
4. Injection and Reversible Inactivation
<ol>
	<li>Set the microinjection pump to inject 400 to 800 nl at 40 nl/min and press Run to start the injection. Note that spike rate will show reduction during the injection. 
	NOTE: In the experimental setup, neural activity recovered within an hour after the end of GABA delivery. Any calibrated mechanical device can be used to apply pressure on the microinjection syringe in order to conduct the injection.</li>
	<li>After acquisition of the electrophysiological data, euthanize using a method approved by the local Animal Ethics Community.</li>
</ol>

<ol><li>Martin, J. H., Ghez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacological+inactivation+in+the+analysis+of+the+central+control+of+movement%5BTitle%5D)+AND+%22Journal+Of+Neuroscience+Methods%22%5BJournal%5D)">Pharmacological inactivation in the analysis of the central control of movement.</a> Journal Of Neuroscience Methods. 86 (2), 145-159 (1999).</li>
<li>Ponce, C. R., Hunter, J. N., Pack, C. C., Lomber, S. G., Born, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contributions+of+indirect+pathways+to+visual+response+properties+in+macaque+middle+temporal+area+MT%5BTitle%5D)+AND+%22The+Journal+Of+Neuroscience+The+Official+Journal+Of+The+Society+For+Neuroscience%22%5BJournal%5D)">Contributions of indirect pathways to visual response properties in macaque middle temporal area MT.</a> The Journal Of Neuroscience The Official Journal Of The Society For Neuroscience. 31 (10), 3894-3903 (2011).</li>
<li>Lomber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+advantages+and+limitations+of+permanent+or+reversible+deactivation+techniques+in+the+assessment+of+neural+function%5BTitle%5D)+AND+%22Journal+Of+Neuroscience+Methods%22%5BJournal%5D)">The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function.</a> Journal Of Neuroscience Methods. 86 (2), 109-117 (1999).</li>
<li>Malpeli, J., Schiller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+of+reversible+inactivation+of+small+regions+of+brain+tissue%5BTitle%5D)+AND+%22Journal+Of+Neuroscience+Methods%22%5BJournal%5D)">A method of reversible inactivation of small regions of brain tissue.</a> Journal Of Neuroscience Methods. 1 (2), 143-151 (1979).</li>
<li>Lomber, S. G., Payne, B. R., Horel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cryoloop%3A+an+adaptable+reversible+cooling+deactivation+method+for+behavioral+or+electrophysiological+assessment+of+neural+function%5BTitle%5D)+AND+%22Journal+Of+Neuroscience+Methods%22%5BJournal%5D)">The cryoloop: an adaptable reversible cooling deactivation method for behavioral or electrophysiological assessment of neural function.</a> Journal Of Neuroscience Methods. 86 (2), 179-194 (1999).</li>
<li>Gonzalez-Perez, O., Guerrero-Cazares, H., Quiñones-Hinojosa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+of+deep+brain+structures+with+microinjections+for+delivery+of+drugs%2C+viral+vectors%2C+or+cell+transplants%5BTitle%5D)+AND+%22Journal+Of+Visualized+Experiments%22%5BJournal%5D)">Targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants.</a> Journal Of Visualized Experiments. (46), (2010).</li>
<li>Hupé, J., Chouvet, G., Bullier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+and+temporal+parameters+of+cortical+inactivation+by+GABA%5BTitle%5D)+AND+%22Journal+Of+Neuroscience+Methods%22%5BJournal%5D)">Spatial and temporal parameters of cortical inactivation by GABA.</a> Journal Of Neuroscience Methods. 86 (2), 129-143 (1999).</li>
<li>Casanova, C., McKinley, P., Molotchnikofff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Responsiveness+of+Reorganized+Primary+Somatosensory+%28SI%29+Cortex+after+Local+Inactivation+of+Normal+SI+Cortex+in+Chronic+Spinal+Cats%5BTitle%5D)+AND+%22Somatosensory+%26+Motor+Research%22%5BJournal%5D)">Responsiveness of Reorganized Primary Somatosensory (SI) Cortex after Local Inactivation of Normal SI Cortex in Chronic Spinal Cats.</a> Somatosensory & Motor Research. 8 (1), 65-76 (1991).</li>
<li>Malpeli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversible+inactivation+of+subcortical+sites+by+drug+injection%5BTitle%5D)+AND+%22Journal+Of+Neuroscience+Methods%22%5BJournal%5D)">Reversible inactivation of subcortical sites by drug injection.</a> Journal Of Neuroscience Methods. 86 (2), 119-128 (1999).</li>
<li>Minville, K., Casanova, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+frequency+processing+in+posteromedial+lateral+suprasylvian+cortex+does+not+depend+on+the+projections+from+the+striate-recipient+zone+of+the+cat%E2%80%99s+lateral+posterior-pulvinar+complex%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Spatial frequency processing in posteromedial lateral suprasylvian cortex does not depend on the projections from the striate-recipient zone of the cat’s lateral posterior-pulvinar complex.</a> Neuroscience. 84 (3), 699-711 (1998).</li>
<li>Diao, Y., Wang, Y., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Representation+of+the+binocular+visual+field+in+the+superior+colliculus+of+the+albino+rat%5BTitle%5D)+AND+%22Experimental+Brain+Research%22%5BJournal%5D)">Representation of the binocular visual field in the superior colliculus of the albino rat.</a> Experimental Brain Research. 52 (1), 67-72 (1983).</li>
</ol>

We have nothing to disclose.

The proposed protocol was designed to solve the challenges arising from current reversible inactivation methods. Specifically, this project aimed at refining the methods used for chemical microinjections of substances modulating neural activity, particularly in deep brain structures. A technical challenge emerging from this type of setup is the need for both probes to be colocalized in the same restricted space in vivo in order to derive precise recordings at the injection site. This issue can be overcome by usi...

The inactivation of cortical areas and sub-cortical nuclei is important in the study of functional relations between various brain structures2-4. Recent literature has employed loss-of-function chemical or cryogenic techniques to study the role of brain structures2,5. In regard to pharmacological microinjections, small volumes of drugs can be administered into a brain region at a controlled rate while minimizing the collateral damage to the surrounding tissue6,7. This technique can be used to deliver specific agonists, inverse agonists or antagonists to study the effect of different pharmacological targets on neuronal activity. Such effects can also be studied by measuring changes in neuronal responses from distant locations, allowing researchers to study the relationships between different cortical and subcortical structures.
Here, we demonstrate the assembly of a device, the injectrode, capable of both recording electrophysiological signals and delivering small amounts of drugs at the target location. We demonstrate the capabilities of this system by injecting GABA, a common inhibitor of neuronal activity, in the rat superior colliculus. This region is sensitive to visual stimulation, which allowed us to use visually evoked multiunit activity&#160;to confirm injectrode localization. The reversibility of the inactivation was assessed by the recovery of normal neuronal activity following the end of GABA injection.
The ability to monitor multi-unit activity from the injection site allows for the fine tuning of the injection rates and volumes needed to achieve the desired pharmacodynamic response. Therefore, an advantage of this technique is the potential limiting of tissue damage caused by microperfusion, since the smallest effective volumes are injected. The proposed protocol provides a cost efficient method for generating the disposable hardware necessary for conducting experiments where drug delivery and local neuronal activity recording is desired.

<table><tbody><tr><td>Injection pump (UltraMicroPump III)</td><td>WPI</td><td>#UMP3</td><td></td></tr><tr><td>Injection console (Micro4 Controller)</td><td>WPI</td><td>#SYS-MICRO4</td><td></td></tr><tr><td>Hamilton syringe</td><td>Hamliton</td><td>(80301) 701LT 10 &amp;micro;L SYR</td><td>Syringes between 5 and 10 &amp;mu;l used</td></tr><tr><td>Flexible plastic adhesive</td><td>Lepage</td><td>393915</td><td>The gel form is easier to apply to the shaft of the 30&amp;nbsp;G hypodermic needle forming a waterproof seal when dry.</td></tr><tr><td>Glass pipettes</td><td>WPI</td><td>#TW100F-4</td><td>Thin wall, 1&amp;nbsp;mm OD, 0.75&amp;nbsp;mm ID with filament pipettes used</td></tr><tr><td>720 Needle Pipette Puller</td><td>Kopf</td><td>720</td><td></td></tr><tr><td>Silver wire</td><td>A-M Systems, Inc.</td><td>782500</td><td>Bare 0.010&amp;rdquo;</td></tr></tbody></table>

simultaneous electrophysiological recording and micro-injections of inhibitory agents in the rodent brain

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

The construction of the injectrode is illustrated in Figure 1. A silver wire (C) is fed into a glass pipette (D) with a portion of the wire bent and protruding out from the opening. A 30 G needle (B) is attached and sealed to the opening of the glass pipette with glue. After the pipette has been filled with the injection substance, a glass micro syringe (A) is attached to the needle. It is important that there is a good seal where the micro syringe connects with the needle (E) and where the silver wire p...

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

The overall goal of this procedure is to construct an affordable inject road to micro inject neurotransmitters into the rat superior colliculus while monitoring multi-unit neuronal activity. This is accomplished by first creating a glass pipette using a glass capillary and a vertical micro pipette puller. The second step is to insert a silver wire into the capillary with a section of wire protruding out from the non tapered end.Next glue is used to create a tight seal between the glass pipette and a hypodermic needle. The final steps are to fill the pipette with a Chicago sky blue colored GABA solution attached to a micro syringe filled with mineral oil and fixed to a micro injector. Ultimately, this construction of an inject with accessible and affordable parts can be used to inactivate deep brain structures while recording neuronal activity of the inactivated area before, during, and after injection of a drug.There are a few advantages of this technique over other existing methods such as gas-based pressure pulses and photic injection systems. One is that it is a cost efficient method that allows for function-based control over the drug delivery, which limits the tissue damage that is caused from over injection. Another advantage that this system offers is a combined recording capability, which allows for efficient control over the temporal component of neural deactivation.To prepare the recording injection pipettes, first pull a seven centimeter capillary with a one millimeter outer diameter for a cop vertical model. Seven 20 or similar unit. Set the heater to 18 and the solenoid to one next, wearing gloves manually break the tip to make an opening and verify under a microscope that the inner diameter is between 30 and 40 micrometers.Measure this with the viewfinders ruler. It will produce a relatively low impedance of 0.3 to 0.6 mega ohms. Then from the non tapered end, insert a silver wire into the pipette about seven centimeters long, and leave one to two centimeters hanging out of the pipette.Bend this excess wire orthogonally to the capillary. Next, take a 30 gauge hypodermic needle and apply flexible plastic adhesive onto the shaft. Then slowly insert the needle into the pipette.As far as possible. Add more adhesive to create a seal at the junction between the pipette and the needle. With the pipette tip upwards, let the assembly dry for about 12 hours.After anesthetizing and putting the rat into a stereotaxic frame, apply ophthalmic ointment shave its head clean. After applying a local anesthetic and confirming a surgical plane of anesthesia, proceed with incising the scalp along the median using a number 10 scalpel blade. This will expose the coronal and sagittal sutures.Then using a surgical spatula, identify the Lambda and bgma anatomical references. Adjust the position of the head so that these landmarks are level in the same horizontal plane. Then zero, a fine rigid pointer like a glass tube on the bgma.Next, adjust the position of the tube to a location of interest using coordinates from the bgma. Use the sharpie to draw a square around the target area for the craniotomy. Now with the surgical sterilized drill, slowly remove the bone along the marks.Begin with using as little pressure as possible and keep the drill moving to avoid head induced tissue damage due to friction when the bone has become thin, carefully remove the square section with tweezers. The Dora MA does not need to be removed as the inject will penetrate it going forward. Keep the exposed cortex irrigated with cerebral spinal fluid.Prepare the inject by first filling of five to 10 microliter syringe with mineral oil. Next, prepare the drug of interest with a dye in physiological saline. Here 0.5%Chicago sky blue is mixed with 300 micromolar gaba.Now fill the injection pipette by first loading a one milliliter syringe with a prepared solution, and then using the syringe to load the pipette. When removing the syringe, keep light pressure on the plunger, so a vacuum does not remove solution from the inject road. Check for escaping fluid at possible leak points.Swab up any excess fluid and recheck for leakage. Now, attach the micros syringe with mineral oil to the inject road. Then wipe away any excess solution.With gauze. Check the injection tip. It should be possible to flow small drops of solution through it, otherwise it is blocked or now securely mount the injector to a micro pump system.Then move the position of the inject road to the target coordinates and lower the electrode to the superior colus, which can be identified through the recording of visual evoked potentials. While the electrode is lowered, apply a small positive pressure by applying a low rate of injection to avoid clogging. Interrupt the injection when the structure is reached.After inserting the inject, cover the exposed cortex with warm agar to prevent desiccation. After the final placement of the electrode, wait 30 minutes to allow the stabilization of the tissue surrounding the electrode. Then inject 400 to 800 nanoliters of solution at a rate of 40 nanoliters per minute until an activation of neuronal activity in the superior colus, the electrode should show a reduction in spikes during the injection of the inhibitory solution.Visually evoked potentials were obtained in the superior colus. Following a 300 millisecond flash to the contralateral eye upon the injection of gaba, the spiking activity in response to a flash stimulus was suppressed for 45 to 60 minutes. While attending this procedure, it is important to check for leaks during loading and to verify that the drug solution is flowing freely through the inject tip.

simultaneous-electrophysiological-recording-micro-injections

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JoVE Journal

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In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

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A Micro-drive Array Method for Electrophysiological Recording from Multiple Brain Regions

This video demonstrates the procedure for concurrent recordings of co-localized electroencephalography (EEG) and local field potentials (LFPs) in an anesthetized rat. The process involves preparing the rat and attaching stimulating electrodes to its whisker pads. An EEG spider electrode is placed on the rat's skull for surface EEG measurements. At the same time, a multi-channel microelectrode is inserted into a specific brain region through a burr hole in the skull to record neural activity...

Concurrent Electroencephalographic and Local Field Potential Recordings in an Anesthetized Rat

Source: Lai, J., et al. Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain. J. Vis. Exp. (2015)This video demonstrates a microinjection-based technique for simultaneous neuronal recording and inhibition in the rat superior colliculus. The procedure involves stereotaxic targeting, skull drilling, and the insertion of an injectrode, which delivers an inhibitory neurotransmitter while recording neuronal activity. Light stimulation evokes...

Inhibition of Neuronal Activity and Its Detection Using Electrophysiology in a Rat

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Microinjectrode System for Combined Drug Infusion and Electrophysiology

Learn to construct a dual-function glass pipette for simultaneous microinjection of GABA and electrophysiological recording in the rat superior colliculus. This affordable method enables real-time monitoring of neuronal activity during targeted drug delivery.

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

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